EGFR targeted nano-drug carrier and preparation method thereof

Abstract

The invention discloses an EGFR (epidermal growth factor receptor) targeted nano-drug carrier and a preparation method thereof, the EGFR targeted nano-drug carrier comprises drug-loading nanoparticles, an erythrocyte membrane coating the drug-loading nanoparticles and GE11 peptide connected to the erythrocyte membrane, and the drug-loading nanoparticles are formed by connecting tannic acid, ellagic acid and 1, 4-phenyldiboronic acid through boric acid ester bonds. The EGFR targeted nano-drug carrier disclosed by the invention can be enriched at an EGFR overexpressed tumor site in a targeted manner, and can be effectively prevented from being cleared by an immune system, and the drug loaded by the drug-loaded nanoparticles is released in a high-active oxygen environment, so that the treatment effect on tumors can be improved, and the toxic and side effects on normal tissues can be reduced; and photothermal therapy and chemotherapy effects can be simultaneously generated under NIR illumination, and the anticancer efficiency can be remarkably improved through the synergistic effect of the photothermal therapy and the chemotherapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to nanomedicine carriers targeting tumors and their preparation methods. Background Technology

[0002] Modern research has demonstrated that ellagic acid (EA), a natural polyphenol, is abundant in Phyllanthus emblica leaves and possesses a wide range of pharmacological effects. It can scavenge free radicals, has antioxidant and anti-aging properties, lowers blood lipids, improves diabetic complications, combats atherosclerosis, protects against liver damage and fibrosis, and exhibits analgesic, anti-inflammatory, anti-mutagenic, anti-teratogenic, and anti-tumor effects. Ellagic acid has been confirmed by numerous studies to have significant antitumor activity. However, its low bioavailability, insufficient systemic exposure, and time-dependent increase in toxicity limit its therapeutic efficacy as an anticancer drug. To overcome these obstacles, an effective delivery system must be established for its use in cancer treatment.

[0003] In tumors, EGFR overexpression leads to sustained activation of signaling pathways, promoting abnormal cell proliferation and is closely related to the development and progression of various malignant tumors, making it an important therapeutic target. EGFR overexpression is observed in approximately 50%–70% of esophageal cancer patients. However, current EGFR-targeted therapies (such as antibodies and tyrosine kinase inhibitors) have limited efficacy. Summary of the Invention

[0004] In view of this, the present invention provides an EGFR-targeted nanomedicine carrier and its preparation method to solve the technical problem of effectively delivering ellagic acid to the tumor region and improving its therapeutic effect.

[0005] The EGFR-targeted nanomedicine carrier of the present invention comprises drug-loaded nanoparticles, a red blood cell membrane encapsulating the drug-loaded nanoparticles, and a GE11 peptide attached to the red blood cell membrane. The drug-loaded nanoparticles are composed of tannic acid, ellagic acid, and 1,4-phenylenediboronic acid linked by borate ester bonds.

[0006] Furthermore, the GE11 peptide is linked to the erythrocyte membrane via a DSPE-PEG bridging molecule.

[0007] This invention also discloses a method for preparing the above-mentioned EGFR-targeted nanomedicine carrier, comprising:

[0008] 1) Preparation of drug-loaded nanoparticles:

[0009] Tannic acid was first dissolved in dimethyl sulfoxide, and then 1,4-phenylenediboric acid and ellagic acid were added to prepare a reaction mixture. The reaction mixture was stirred at room temperature to complete the reaction, and then the reaction mixture was dialyzed in water to remove unreacted substances. Finally, the product was collected and dried to obtain drug-loaded nanoparticles.

[0010] 2) Preparation of GE11 peptide-modified erythrocyte membranes:

[0011] Red blood cell membranes were gently incubated with DSPE-PEG-GE11 at 37°C with gentle shaking, and GE11-RBCM was obtained by embedding DSPE into the lipid layer of red blood cell membranes.

[0012] 3) Drug-loaded nanoparticles were encapsulated with GE11-RBCM.

[0013] Furthermore, the DSPE-PEG-GE11 mentioned in step 2) is prepared by the following method:

[0014] The GE11 peptide was dissolved in a dimethyl sulfoxide solution containing DSPE-PEG-MAL to prepare a reaction mixture, and then the reaction mixture was allowed to complete the reaction at room temperature. After the reaction was completed, the reaction mixture was transferred to a dialysis bag and dialyzed in water to remove the solvent and excess GE11 peptide. The aqueous solution of the product was freeze-dried to obtain DSPE-PEG-GE11.

[0015] Further, step 3) involves encapsulating drug-loaded nanoparticles with GE11-RBCM, which includes mixing GE11-RBCM with drug-loaded nanoparticles in an ultrasonic bath and then performing an extrusion operation to obtain GE11-RBCM@drug-loaded nanoparticles.

[0016] The beneficial effects of this invention are:

[0017] 1. In this invention, the EGFR-targeted nanomedicine carrier loads ellagic acid into drug-loaded nanoparticles. The drug-loaded nanoparticles utilize the ability of the GE11 peptide to specifically bind to EGFR, thereby preferentially enriching the drug-loaded nanoparticles in tumor sites overexpressing EGFR, which can significantly increase the drug concentration in the tumor site.

[0018] 2. In this invention, the EGFR-targeted nanomedicine carrier imparts a "self-like" appearance to the drug-loaded nanoparticles through the red blood cell membrane shell, which can effectively prevent them from being cleared by the immune system and significantly prolong the biological half-life of the drug-loaded nanoparticles.

[0019] 3. In this invention, the EGFR-targeted nanomedicine carrier has borate ester bonds that connect the drug-loaded nanoparticles. These bonds can break and decompose in the highly reactive oxygen environment of tumor tissue, thereby triggering the release of ellagic acid from the drug-loaded nanoparticles. This allows ellagic acid to be released mainly in tumor tissue, which can improve the therapeutic effect on tumors and reduce the toxic side effects on normal tissues.

[0020] 4. In this invention, the EGFR-targeted nanomedicine carrier contains tannic acid in its drug-loaded nanoparticles, which can rapidly generate heat and rise in temperature under near-infrared light irradiation at a wavelength of 808nm, thereby achieving photothermal therapy on tumor tissue.

[0021] Furthermore, the heat generated by tannic acid promotes the breaking of boronic acid ester bonds with redox properties, accelerating the release of the chemotherapy drug ellagic acid from drug-loaded nanoparticles. The specific process includes oxidative rearrangement: ROS reacts with boron atoms in the boronic acid ester bond, leading to the rearrangement of the boronic acid ester structure; hydrolysis reaction: the rearranged intermediate is further hydrolyzed to generate phenolic hydroxyl groups; elimination reaction: the phenolic hydroxyl groups break the boronic acid ester bond through a 1,6-elimination reaction, thus achieving a response to ROS.

[0022] Therefore, this EGFR-targeted nanomedicine carrier can simultaneously produce photothermal therapy and chemotherapy effects, overcoming the limitations of simple photothermal or chemotherapy alone. The synergistic effect of the two can significantly improve the anti-cancer efficiency. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of GE11-modified red blood cell membranes encapsulating drug-loaded nanoparticles.

[0024] Figure 2 This describes the synthesis pathway for drug-loaded nanoparticles.

[0025] Figure 3 MALDI-TOF-MS spectrum of drug-loaded nanoparticles after synthesis.

[0026] Figure 4 The synthetic route for DSPE-PEG-GE11 is shown.

[0027] Figure 5 Analysis of the 1H NMR hydrogen spectrum of DSPE-PEG-GE11.

[0028] Figure 6 For flight mass spectrometry detection (MALDI-TOF-MS spectrum) of DSPE-PEG-GE11.

[0029] Figure 7 Characterization diagrams of EGFR-targeted nanomedicine carriers after preparation, including preparation of drug-loaded nanoparticles, erythrocyte membrane coating, and GE11 peptide conjugation.

[0030] Figure 8 To assess the ROS-responsive properties of EGFR-targeted nanomedicine carriers, borate ester bonds that can be broken by reactive oxygen species are introduced into the drug-loaded nanoparticle structure. When the nanoparticles enter the high-ROS tumor microenvironment, these bonds break, accelerating the decomposition of the drug-loaded nanoparticles and the release of the drug.

[0031] Figure 9 The photothermal properties of EGFR-targeted nanomedicine carriers.

[0032] Figure 10 EGFR-targeted nanomedicine carriers promote necrosis and apoptosis in tumor cells.

[0033] Figure 11 To enable EGFR-targeted nanomedicine carriers to produce a long retention effect in vivo.

[0034] Figure 12 To achieve the synergistic effect of photothermal and chemotherapy on tumors by EGFR-targeted nanomedicine carriers.

[0035] Figure 13 To ensure the biosafety of EGFR-targeted nanomedicine carriers. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0037] Example 1: In this example, the EGFR-targeted nanomedicine carrier includes drug-loaded nanoparticles, a red blood cell membrane coating the drug-loaded nanoparticles, and a GE11 peptide attached to the red blood cell membrane. The drug-loaded nanoparticles are composed of tannic acid, ellagic acid, and 1,4-phenylenediboric acid linked by borate ester bonds. In this example, the GE11 peptide is attached to the red blood cell membrane via a DSPE-PEG bridging molecule.

[0038] Example 2: A method for preparing the EGFR-targeted nanomedicine carrier, comprising:

[0039] 1) Preparation of drug-loaded nanoparticles:

[0040] First, 170.1 mg of tannic acid (TA) was dissolved in dimethyl sulfoxide (DMSO), and then 165.75 mg of 1,4-phenylenediboric acid (PBA) and 15 mg of ellagic acid (EA) were added to prepare a reaction mixture. The reaction mixture was stirred at room temperature for 24 h to complete the reaction. Then, the reaction mixture was dialyzed in water (dialysis membrane molecular weight cutoff: 500 Da) to remove unreacted EA and DMSO. Finally, the product was collected and dried to obtain drug-loaded nanoparticles in solid form.

[0041] 2) Preparation of GE11 peptide-modified erythrocyte membranes:

[0042] Red blood cell membranes were incubated with DSPE-PEG-GE11 at 37°C with gentle shaking for 4 hours, and GE11-RBCM was obtained by embedding DSPE into the lipid layer of red blood cell membranes.

[0043] The DSPE-PEG-GE11 mentioned in this step is prepared by the following method:

[0044] 100 mg of GE11 (YHWYGYTPQNVI‐SH) peptide was dissolved in a dimethyl sulfoxide solution containing 300 mg of DSPE-PEG-MAL to prepare a reaction mixture, and then the reaction mixture was allowed to complete the reaction at room temperature. After the reaction was completed, the reaction mixture was transferred to a dialysis bag with a molecular weight cutoff of 2 kDa and dialyzed in water to remove the solvent and excess GE11 peptide. The aqueous solution of the product was freeze-dried to obtain DSPE-PEG-GE11.

[0045] The erythrocyte membrane described in this embodiment is prepared by the following method:

[0046] Blood was collected from the orbital sinus of 8-week-old C57 mice. The blood was centrifuged at 3500 rpm for 5 min at 4°C, and the supernatant serum was carefully removed. The collected red blood cells were washed three times with phosphate-buffered saline (PBS, pH = 7.4) to remove residual serum and impurities. The washed red blood cells were resuspended in 0.25×PBS (pH = 7.4) and incubated at 4°C for 20 min to induce cell membrane rupture using a hypotonic solution. Subsequently, the red blood cell suspension was centrifuged at 10000 rpm for 5 min at 4°C, and the supernatant was collected. After washing with PBS and repeating the centrifugation three times, the pink precipitate was collected as the red blood cell membrane (RBCM). The RBCM was resuspended in ultrapure water and sonicated. The treated RBCM was stored at -80°C.

[0047] 3) Encapsulating drug-loaded nanoparticles with GE11-RBCM. The specific operation includes: mixing GE11-RBCM with drug-loaded nanoparticles in an ultrasonic bath, and then extruding to obtain GE11-RBCM@drug-loaded nanoparticles.

[0048] The performance of the EGFR-targeted nanomedicine carriers in the above embodiments was tested as follows:

[0049] In vitro photothermal performance testing: 1 mL of ellagic acid (EA), tannic acid (TA), drug-loaded nanoparticles (NPs), or EGFR-targeted nanomedicine carriers (GE11-RBCM@NPs) was irradiated with an 808 nm laser (power density of 2 W / cm², irradiation time 0-5 min). Temperature changes were recorded using an infrared thermal imager and analyzed using FLIR Tools software. The photothermal stability of the materials was evaluated through 5 irradiation-cooling cycles. The figures show that the EGFR-targeted nanomedicine carrier exhibits good photothermal properties, rapidly heating up under near-infrared light irradiation to generate photothermal benefits.

[0050] In vitro toxicity and antitumor effects of GE11-RBCM-coated nanoparticles (GE11-RBCM@NPs): 1) Cellular uptake: To evaluate the targeting effect of GE11 peptide on the esophageal cancer cell line mEC25, the cellular uptake efficiency of drug-loaded nanoparticles (NPs), erythrocyte membrane-coated drug-loaded nanoparticles (RBCM@NPs), and EGFR-targeted nanomedicine carriers (GE11-RBCM@TPE NPs) was tracked. Specifically, drug-loaded nanoparticles (NPs) were first labeled with the lipophilic fluorescent dye DiD, and then unloaded DiD was removed by water dialysis to obtain NPs@DiD. Next, RBCM and GE11-RBCM were co-extruded onto the surface of NPs@DiD to prepare RBCM@NPs@DiD and GE11-RBCM@NPs@DiD, respectively. Fluorescence microscopy and flow cytometry (FCM) were used to evaluate the uptake of NPs@DiD, RBCM@NPs@DiD, and GE11-RBCM@NPs@DiD by mEC25 cells. Solutions of the three nanoparticles at a concentration of 150 μg / mL were added to each well containing 1×10⁻⁶ nanoparticles. 5 Cells were incubated in 6-well plates at densities of 1000 cells / well for 0.5 hours, 1 hour, and 2 hours, respectively. Cells were then observed under a fluorescence microscope. Simultaneously, cellular uptake was quantitatively analyzed using FCM. 2) In vitro blood compatibility test: The in vitro blood compatibility of GE11-RBCM@NPs was determined using a direct contact method. Specifically, 0.1 mL of whole blood was added to different concentrations (EA concentration ranged from 20 μg / mL to 80 μg / mL) of GE11-RBCM@NPs solution. The absorbance of the supernatant was then measured. 0.09% sodium chloride solution and distilled water were used as negative and positive controls, respectively. 3) Cytotoxicity test: The anticancer activity of GE11-RBCM@NPs against mEC25 cells was evaluated using the CCK-8 cell viability assay. First, cells were incubated at a density of 1×1000 mEC25 cells / well. 410 mEC25 cells were seeded in 96-well plates and incubated at 37°C for 24 hours. Then, the original medium was replaced with 200 μL of fresh medium containing different drug formulations (free EA, NPs, RBCM@NPs, GE11-RBCM@NPs, and GE11-RBCM@NPs + NIR) (corresponding to EA concentrations ranging from 1 μg / mL to 64 μg / mL), and incubation continued for another 24 hours. Subsequently, 20 μL of CCK-8 reagent was added to each well, and incubation continued for another 4 hours. Finally, the absorbance of each solution was measured at 490 nm using a microplate reader to calculate the cell viability of each group. Simultaneously, the data for each group were fitted to obtain the half-maximal inhibitory concentration (IC50) for comparison. 4) Clonogenesis assay: 2.5 × 10⁶ mEC25 cells were seeded in 96-well plates. 6 Cells were seeded at a density of 10 cells / mL in culture dishes and allowed to adhere for 24 hours. Cells were then treated with free EA, NPs, RBCM@NPs, GE11-RBCM@NPs, and GE11-RBCM@NPs+NIR (corresponding to an EA concentration of 32 μg / mL) for 24 hours. Untreated cells served as a blank control. After treatment, cells were collected, washed with phosphate-buffered saline (PBS), and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in sterile PBS. Cell counts were performed under a microscope, and cell concentrations were adjusted. Cells were seeded at a density of 500 cells / well in 6-well plates, with 3 parallel wells per group. The culture medium was changed every 3 days, and cultured for 10 to 14 days until clear cell clones were observed, at which point culture was stopped. The culture medium was discarded, and the cells were washed three times with PBS. Add approximately 1 mL of 4% paraformaldehyde to each well to fix cells for 30 minutes, then add 1 mL of crystal violet solution for staining for 30 minutes. Wash with PBS 3-4 times, photograph the cells, and count the number of clones. 5) Dead / live cell detection: Cells at a density of 4 × 10⁶ cells / well... 5 mEC25 cells were seeded at a density of 10 cells / well in 6-well plates and incubated for 24 hours. Subsequently, mEC25 cells were treated for 24 hours with culture systems containing free EA, NPs, RBCM@NPs, GE11-RBCM@NPs, and GE11-RBCM@NPs + NIR (corresponding to EA concentration of 32 μg / mL). After treatment, cells were collected and washed with PBS. Cells were stained using a live / dead cell staining kit. 6) Apoptosis detection: Cells were seeded at a density of 5.0 × 10⁶ cells / well. 5mEC25 cells were seeded at 1 / well in 6-well plates and incubated overnight. Then, cells were treated for 24 hours with the same concentrations of free EA, NPs, RBCM@NPs, GE11-RBCM@NPs, and GE11-RBCM@NPs+NIR (corresponding to EA concentration of 32 μg / mL). Untreated mEC25 cells served as a control. To quantitatively analyze the apoptosis rate, treated cells were collected after trypsin digestion, washed twice with PBS, and resuspended in PBS. Subsequently, cells were stained and analyzed using the Annexin V-FITC apoptosis detection kit. Results are shown below. Figure 10 As shown, experiments have demonstrated that EGFR-targeted nanomedicine carriers can promote necrosis and apoptosis of tumor cells.

[0051] In vivo photothermal performance testing: GE11-RBCM@NPs formulation (concentration 2 mg / mL, injection volume 0.2 mL) was injected intravenously into the tail vein of tumor-bearing mice. 24 h after injection, the tumor site was irradiated with an 808 nm laser (power density 2 W / cm², irradiation for 5 min), and the tumor temperature was recorded simultaneously. Results are as follows: Figure 9 As shown, GE11-RBCM@NPs can rapidly heat up under near-infrared light irradiation, generating photothermal benefits.

[0052] In vivo circulation and tumor targeting: Male C57BL / 6 mice (approximately 25 g in weight) were used in the experiment. When constructing the orthotopic esophageal cancer model, 1×10⁻⁶ mice were used... 6 Mouse esophageal cancer cells (mEC25) were mixed with matrix gel at a volume ratio of 1:10 and injected into the esophageal wall of mice. In the in vivo circulation study, DiD-labeled drug-loaded nanoparticles (NPs@DiD) or GE11-RBCM@NPs@DiD (concentration 2 mg / mL, injection volume 200 μL) were injected via tail vein. Blood samples were collected at predetermined time points, and the fluorescence intensity of the samples was detected to analyze the circulation of nanoparticles in the blood. In the tumor targeting study, free DiD, NPs@DiD, erythrocyte membrane-coated DiD-labeled drug-loaded nanoparticles (RBCM@NPs@DiD), and GE11-RBCM@NPs@DiD were injected via tail vein, respectively. 24 h after injection, the mice were perfused with 4% paraformaldehyde, and then the tumor tissue was excised. Two-dimensional / three-dimensional (2D / 3D) fluorescence imaging analysis was performed to evaluate the tumor targeting ability of the nanoparticles. The results are as follows: Figure 11 As shown, EGFR-targeting nanomedicine carriers can produce a long retention effect in vivo, avoiding being cleared by the body, and can actively target EGFR to accurately identify esophageal cancer cells, thus achieving precision treatment.

[0053] In vivo antitumor efficacy and safety: A concentration of 5×10⁻⁶ was used. 7 0.2 mL of mEC25 cell suspension ( / mL) was subcutaneously injected into the hind limbs of mice. When the tumor volume grew to approximately 100 mm³, the mice were randomly divided into groups and treated via tail vein injection with PBS, free EA, NPs, RBCM@NPs, GE11-RBCM@NPs, or GE11-RBCM@NPs + near-infrared light (NIR) (injected every two days, n = 5 per group). Tumor volume and mouse weight changes were recorded regularly. On day 14 of the experiment, mice were sacrificed, and tumor and major organ tissues were collected for hematoxylin-eosin staining (H&E staining), TUNEL staining mediated by terminal deoxyribonucleotide transferase (TUNEL), and immunohistochemical staining (detecting indicators including Ki67, epidermal growth factor receptor (EGFR), platelet endothelial cell adhesion molecule-31 (CD31), and caspase-3). Simultaneously, blood alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CREA) levels were measured to assess drug toxicity to the liver and kidneys. Experimental results are as follows: Figure 12-13 As shown, EGFR-targeted nanomedicine carriers significantly enhance anti-tumor effects through the synergistic effect of photothermal and chemotherapy on tumors; moreover, EGFR-targeted nanomedicine carriers have good biosafety and do not damage the function of the liver and kidneys or major organs such as the liver, spleen, lungs and kidneys.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An EGFR-targeted nanomedicine carrier, characterized in that: It includes drug-loaded nanoparticles, a red blood cell membrane encapsulating the drug-loaded nanoparticles, and a GE11 peptide attached to the red blood cell membrane. The drug-loaded nanoparticles are composed of tannic acid, ellagic acid, and 1,4-phenylenediboronic acid linked by borate ester bonds.

2. The EGFR-targeted nanomedicine carrier according to claim 1, characterized in that: The GE11 peptide is linked to the erythrocyte membrane via a DSPE-PEG bridging molecule.

3. The method for preparing the EGFR-targeted nanomedicine carrier as described in claim 1, characterized in that: include: 1) Preparation of drug-loaded nanoparticles: Tannic acid was first dissolved in dimethyl sulfoxide, and then 1,4-phenylenediboric acid and ellagic acid were added to prepare a reaction mixture. The reaction mixture was stirred at room temperature to complete the reaction, and then the reaction mixture was dialyzed in water to remove unreacted substances. Finally, the product was collected and dried to obtain drug-loaded nanoparticles. 2) Preparation of GE11 peptide-modified erythrocyte membranes: Red blood cell membranes were gently incubated with DSPE-PEG-GE11 at 37°C with gentle shaking, and GE11-RBCM was obtained by embedding DSPE into the lipid layer of red blood cell membranes. 3) Drug-loaded nanoparticles were encapsulated with GE11-RBCM.

4. The method for preparing EGFR-targeted nanomedicine carriers according to claim 3, characterized in that: The DSPE-PEG-GE11 mentioned in step 2) is prepared by the following method: The GE11 peptide was dissolved in a dimethyl sulfoxide solution containing DSPE-PEG-MAL to prepare a reaction mixture, and then the reaction mixture was allowed to complete the reaction at room temperature. After the reaction was completed, the reaction mixture was transferred to a dialysis bag and dialyzed in water to remove the solvent and excess GE11 peptide. The aqueous solution of the product was freeze-dried to obtain DSPE-PEG-GE11.

5. The method for preparing EGFR-targeted nanomedicine carriers according to claim 3, characterized in that: Step 3) The process of encapsulating drug-loaded nanoparticles with GE11-RBCM includes: mixing GE11-RBCM with drug-loaded nanoparticles in an ultrasonic bath, and then performing an extrusion operation to obtain GE11-RBCM@drug-loaded nanoparticles.

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