P-QM-1h loaded cetuximab modified nanostructure lipid carrier as well as preparation and application thereof

Abstract

The invention relates to a cetuximab modified nanostructure lipid carrier loaded with p-QM-1h as well as preparation and application of the cetuximab modified nanostructure lipid carrier. A nanoliposome loaded with a therapeutic active drug p-QM-1h is covalently coupled with cetuximab CTX of a high-expression EGFR (epidermal growth factor receptor) in a targeted tumor; the economic cost is low, the encapsulation efficiency is relatively high, and the solubility of p-QM-1h is effectively improved; the nano-particles are uniform, good in dispersion and stability and good in anti-tumor activity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulations or drug delivery, and specifically relates to a loading device. p -QM-1h Cetuximab Modified Nanostructure Lipid Carrier and Its Preparation and Application Background Technology

[0002] In 2022, nearly 20 million new cancer cases were diagnosed globally, and 9.7 million people died from cancer. Due to the aging global population and changing lifestyles, the number of newly diagnosed cancer cases worldwide is projected to reach 35 million by 2050. Furthermore, most cancer patients are diagnosed at an advanced or metastatic stage, and current treatment options include surgery, chemotherapy, and a few targeted and immunotherapies. With advancements in medical science, molecular targeted therapy has propelled cancer treatment into a new phase.

[0003] p-Methylenebenzoquinone ( p -QM) is a class of bioactive compounds with antiviral, antifungal, antibacterial, anti-inflammatory, and antioxidant activities. In our previous work, we... p -QM conducted a series of structural designs and studied their structure-activity relationships. They found that derivatives with meta-substitution exhibited good anti-inflammatory activity. Among them, derivatives substituted with trifluoromethyl groups showed particularly good anti-inflammatory activity. p -QM-1i exerts anti-inflammatory effects in inflammatory cell models and mouse models of ulcerative colitis through the TLR4 / NFB signaling pathway. Meanwhile, derivatives with ortho-substituted morphologies exhibit good anti-cancer activity, especially those with ortho-trifluoromethyl substituted morphologies. p -QM-1h exerts antiproliferative activity in A549 cells by inhibiting thioredoxin reductase TrxR, and exhibits anticancer activity in mice carrying A549 tumors because... p -QM-1h contains a Michael receptor unit; and it has a certain degree of safety in vivo. However, p- QM-1h is a small organic molecule with low solubility in the physiological environment, high dosage, and is not a targeted anti-tumor drug.

[0004] Nanomedicine delivery system loads drugs into nanomaterials and delivers them to target tissues through specific pathways, which can improve the solubility, stability and targeting of drugs, thereby improving the efficacy and reducing side effects. Nanoscale lipid carriers (NLC) are composed of solid lipids and liquid lipids. The addition of liquid lipids can improve the drug loading capacity and be suitable for the delivery of poorly soluble drugs. Immunoliposomes are liposomes with ligands coupled to the surface, which can be used to recognize and bind to specific receptors on cells, thereby achieving targeted delivery. Epidermal growth factor receptor (EGFR) is located on the surface of the cell membrane and is highly or abnormally expressed in malignant tumors. It is related to the inhibition of tumor cell proliferation, angiogenesis, invasion, metastasis and apoptosis. Cetuximab (CTX) is a monoclonal antibody targeting EGFR, which inhibits the EGFR signaling pathway by binding to EGFR, thereby inhibiting tumor growth and proliferation, and plays an important role in cancer treatment. Immunoliposomes coupled with CTX can target tumor cells overexpressing EGFR and achieve effective drug delivery. SUMMARY

[0005] The purpose of the present application is to provide an immunonanostructured liposome with active targeting of EGFR, which loads a therapeutically active drug p The nanoliposome of QM-1h is covalently coupled with cetuximab CTX targeting EGFR highly expressed in tumors; wherein the therapeutically active drug p The structure of QM-1i is as follows: ; The nanoliposome comprises solid liposomes and liquid liposomes, wherein the solid liposomes are DSPC, Cholesterol, DSPE-PEG2000 and DSPE-PEG2000-MAL, and the liquid liposomes are one of Miglyol 812N, medium-chain triglyceride MCT, castor oil or oleic acid.

[0006] Preferably, the liquid liposome is Miglyol 812N.

[0007] Further, the molar ratio of the preferred DSPC, Cholesterol, DSPE-PEG2000 and DSPE-PEG2000-MAL is: 1-4000: 1-4000: 1-600: 1-30, and the molar ratio is preferably 3563: 3563: 490: 9.

[0008] Further, the molar ratio of the solid liposomes and the liquid liposomes is 2000-8000: 2000-4000; preferably, the molar ratio is: 7625: 2375.

[0009] Furthermore, the loaded therapeutically active drug p The nanoliposomes of -QM-1h are spherical with an average particle size ranging from 120nM to 180nM, preferably 150.53±0.68nM, and an encapsulation efficiency (EE) of 85% to 98%, preferably 87.98±3.68%.

[0010] Preferably, the thiolated CTX is covalently coupled to the maleimide site on the surface of the immune nanostructure liposome NLC; the thiolated CTX is generated by the reaction of the cetuximab CTX with Traut.

[0011] Further preferably, the molar ratio of cetuximab (CTX) to solid liposomes DSPE-PEG2000-MAL is 1-15:1, more preferably, the molar ratio is 1-10:1, more preferably, the molar ratio is 1-3:1, and the most preferred molar ratio is 1.5:1.

[0012] A second aspect of this invention provides a method for preparing the aforementioned immune nanostructured liposomes with active EGFR targeting, comprising the following steps: (1) Loading therapeutically active drugs p Preparation of -QM-1h nanoliposomes Solid liposomes DSPC, Cholesterol, DSPE-PEG2000, and DSPE-PEG2000-MAL were dissolved in an organic solvent; under heating conditions, the active substances were... p -QM-1h was dissolved in liquid liposomes; the solid liposome solution and the liquid liposome solution were mixed, and the organic solvent was removed by vacuum distillation; then PBS solution was added for hydration, the liposomes were ultrasonically broken, and the particle size was controlled by extrusion with a polycarbonate membrane to obtain the drug-loaded nanostructured lipid carrier. (2) Antibody-modified drug-loaded nanostructured liposomes CTX- p Preparation of QM-1h-NLC CTX and Traut reagent were mixed in PBS. The sulfurized CTX was dialyzed in PBS to remove excess Traut reagent. The sulfurized CTX was then mixed with the drug-loaded nanostructured lipids prepared in step (1). p Incubate with the QM-1h-NLC vector, then dialyze to remove unbound thiolated CTX to obtain the antibody-modified drug-loaded nanostructured liposomes.

[0013] Preferably, in step (1), the organic solvent is selected from methanol, the PBS solution is 10 mM, pH 7.4, and the polycarbonate membrane is extruded through a 200 nM pore size; In step (2), the molar ratio of CTX to Traut reagent is 1:20, and the sulfurized CTX and...p The molar ratio of -QM-1h-NLC is 1.5:1.

[0014] In a third aspect, the present invention provides the application of the aforementioned immune nanostructured liposomes with active EGFR targeting in the preparation of drugs for treating tumor diseases.

[0015] More preferably, the tumor disease is breast cancer.

[0016] The following is an explanation and description of the terminology used in this invention: The present invention p -QM-1i has the following structure: .

[0017] Cetuximab is a targeted therapy drug that belongs to the epidermal growth factor receptor (EGFR) inhibitor class. It inhibits the growth, proliferation, and metastasis of tumor cells by specifically binding to EGFR and blocking its signal transduction.

[0018] DSPC stands for distearylphosphatidylethanolamine; DSPE-PEG2000-MAL stands for distearylphosphatidylethanolamine-polyethylene glycol 2000-maleimide.

[0019] Obviously, based on the above description of the present invention, and in accordance with common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0020] Beneficial technical effects of the present invention: 1. In order to improve p This invention utilizes NLC-loaded NM-1h to enhance bioavailability and active targeting, reducing dosage and toxicity. p -QM-1h ( p -QM-1h-NLC), and further modified with CTX. p -QM-1h-NLC surface (CTX- p -QM-1h-NLC), used to target EGFR, which is highly expressed in tumor cells. This invention explores CTX- p Preparation method, nanoscale properties, antitumor cell proliferation activity, in vivo antitumor activity in mice, and active targeting of QM-1h-NLC.

[0021] 2. Nanostructured lipid carriers (NLCs) can effectively increase the solubility of poorly soluble drugs. p-QM-1h-NLC); its surface modified with cetuximab (CTX) enables nanomedicine to target and bind to the epidermal growth factor receptor highly expressed on the surface of tumor cells, achieving effective drug delivery (CTX- p- QM-1h-NLC).

[0022] 3. CTX- p- The construction of QM-1h-NLC uses Miglyol 812N as a liquid lipid, which is cost-effective and has a high encapsulation efficiency, effectively improving... p- The solubility of QM-1h; the nanoparticles are uniform, well dispersed, and have good stability; CTX has good solubility in water. p- The modification of QM-1h-NLC exhibits high ligation efficiency while ensuring antibody integrity; CTX- p- The design of QM-1h-NLC does not affect - p- QM-1h exhibits anti-cancer cell proliferation activity. Furthermore, in vivo anti-tumor experiments in mice have shown that CTX- p -QM-1h-NLC effectively inhibited tumor growth in mice in a dose-dependent manner; and 2 mg / kg CTX- p The tumor-inhibiting effect of QM-1h-NLC is comparable to that of 8 mg / kg free drug. p Similar to -QM-1h, effectively improved p -QM-1h's efficacy and reduction p -QM-1h dosage. Attached Figure Description

[0023] Figure 1 : p -Stability testing of QM-1h-NLC; Figure 2 : Figure 2 A is CTX- p Representative transmission electron micrographs of QM-1h-NLC, Figure 2 B represents CTX primary protein structure analysis. Figure 2 C represents tertiary protein structure analysis. Figure 2 D is CTX- p Drug release profile of QM-1h-NLC.

[0024] Figure 3 CTX- p In vivo antitumor activity study of QM-1h-NLC (n=5); Figure 3 A. Tumor volume change curve; Figure 3 B. Tumor weight statistics; Figure 3 C-type tumor morphology diagram; Figure 3 Typical D-type tumor tissue pathology diagram; Detailed Implementation

[0025] The present invention is further illustrated by the following examples, but these are not intended to limit the invention.

[0026] p -QM-1h-NLC and CTX- p The measurement of the nanoscale features of QM-1h-NLC is as follows: Measured using Zetasizer Nano ZSE (Malvern Panalytical) p -QM-1h-NLC and CTX- p Nanoparticle size, polydispersity index (PDI), and zeta potential of -QM-1h-NLC, and p -Stability of QM-1h-NLC.

[0027] 50 L p -QM-1h-NLC and 10 L dimethyl sulfoxide and 20 Mix with LTriton X100, then vortex for 30 seconds and let stand for 30 minutes. Add 320 to the above solution. Add acetonitrile, then vortex the mixture and centrifuge at 12000g for 10 minutes. Collect the supernatant and determine its composition using high-performance liquid chromatography (HPLC). p The content of -QM-1h was used to calculate the encapsulation efficiency (EE) and drug loading (DLC).

[0028] EE% = (Encapsulated in nanoparticles) p -QM-1h quantity / total p -QM-1h dose) x 100% DLC% = (encapsulated in nanoparticles) p -QM-1h volume / total liposome mass) x 100% Example 1: p Preparation and Nanoscale Properties of -QM-1h Based on the mass of each lipid in Table 1, a batch was prepared. p -QM-1h-NLC. 6mg p -QM-1h dissolved in 12.73 mg Miglyol 812N at 75°C. o C. Dissolve the other lipids in 5 mL of methanol, then... p Mix -QM-1h Miglyol 812N solution. Place the mixture in a 100mL bottom flask and incubate at -0.01MPa, 80rpm and 75 oC was subjected to vacuum distillation to remove methanol. The lipid membrane on the flask wall was placed in 2 mL of PBS and incubated at 80 rpm and 75 rpm. o Hydrate at -0.01 MPa for 20 minutes under C conditions, followed by sonication. Then, use an ultrasonic cell disruptor at 25 kHz and 4... o The mixture of 2 mL was broken down at C for 6 minutes. After extrusion through a 200 nm polycarbonate membrane using a liposome extruder, the resulting lipid solution was dialyzed in PBS (10 mM, pH 7.4) for 12 hours (molecular weight cutoff 1000 Da).

[0029] Table 1 Load p Lipid components of -QM-1h nanostructured lipid carriers (NLCs) ;

[0030] p -QM-1h is a small organic molecule with low solubility, which is one of the challenges faced by the pharmaceutical industry due to its poor water solubility and low bioavailability. Based on the lipid ratios in Table 1, a thin-film dispersion method was used to... p -QM-1h is loaded into the NLC to improve p -QM-1h solubility Table 2 p Solubility of QM-1h in PBS or NLC containing different liquid lipids ;

[0031] As shown in Table 2, free p The solubility of QM-1h in phosphate-buffered saline (PBS) is only 14.54 ± 0.60 µg / mL, while the free... p Loading QM-1h into NLC significantly improved its solubility (87.98 ± 3.68 µg / mL) (Table 4), which is beneficial for improving bioavailability and reducing intravenous injection volume in animal experiments. In this study, Miglyol 812N was used as the liquid lipid for the preparation of NLC.

[0032] Table 3. Preparations using different liquid lipids p Comparison of particle size, PDI, Zeta potential and EE% of -QM-1h NLC ;

[0033] Precipitation exists in NLC solutions prepared using oleic acid or castor oil as liquid lipids. As shown in Table 3, besides Miglyol 812N, medium-chain triglycerides (MCTs) and castor oil also exhibit high encapsulation efficiency (EE). However, MCTs have a higher procurement cost, while NLC solutions prepared using castor oil as the liquid lipid have a lower encapsulation efficiency (EE) of 4%. o It will precipitate after being stored at C for 24 hours.

[0034] Table 4 p Features of QM-1h-NLC and CTX-1h-CTX-NLC ;

[0035] The experiment determined p Nanoscale features of QM-1h-NLC (Table 4). p- The QM-1h-NLC particles had a size of 136.77 ± 0.52 nm, a PDI of 0.125 ± 0.001, and a Zeta potential of -20.07 ± 0.67 mV, indicating that the nanoparticles were uniform, well-dispersed, and possessed moderate stability. Furthermore, p The encapsulation efficiency of -QM-1h-NLC was 82.80±1.48%, and the drug loading (DLC) was 7.37±0.13%, indicating that... p The QM-1h-NLC provides sufficient space for efficient loading. p -QM-1h. Furthermore, after 14 days of stability testing, p The particle size, PDI, and Zeta potential of -QM-1h-NLC remained essentially unchanged, indicating good stability. Figure 1 ).

[0036] Example 2: CTX- p Preparation and Nanoscale Properties of QM-1h-NLC CTX antibody (1.0 molar equivalent) was mixed with Traut reagent (2-mercaptoethylamine, 20 molar equivalent) and incubated at 4 °C. o The free amino groups in PBS (10 mM, pH 8.0) were sulfidated at C. The sulfidated CTX antibody was then subjected to 4... o Dialyze overnight at C20°C (MWCO 1000 Da) to remove excess Traut reagent from PBS (10 mM, pH 7.4). The thiol groups (1.5 molar equivalents) of the sulfidated CTX antibody are then reacted with... p Maleimide (1.0 molar equivalent) on QM-1h-NLC at 4 o The reaction was carried out at C for 24 hours. The obtained CTX- p -QM-1h-NLC was dialyzed in PBS (10mM, pH 7.4) for 12 hours to remove excess thiolated CTX antibody (MWCO 3000Da).

[0037] p -QM-1h is a non-selective anti-tumor drug. In order to achieve active targeting of tumors, it will... p The surface of -QM-1h-NLC can be coupled to CTX, which is highly expressed in tumors by EGFR. CTX reacts with Traut to generate thiolized CTX. The thiol group (SH) of the thiolized CTX interacts with the maleimide site retained on the surface of the NLC, which is derived from DSPE-PEG2000-Mal with a total lipid molar ratio of 0.1%.

[0038] According to tests, CTX- p The particle size of -QM-1h-NLC was 150.53±0.68 nm, the polydispersity index (PDI) was 0.123±0.012, and the zeta potential was -19.07±0.25 mV (see Table 4). CTX- p -QM-1h-NLC nanoparticles are uniform, well-dispersed, and stable; CTX- p -QM-1h-NLC has a particle size slightly larger than p -QM-1h-NLC. Further transmission electron microscopy (TEM) analysis showed that the nanoparticles were almost spherical, with particle sizes similar to those obtained from dynamic light scattering (DLS).

[0039] Example 3 CTX- p Determination of QM-1h-NLC antibody characteristics The protein concentration of CTX was determined by using a BCA assay kit to identify the role of CTX in CTX- p -QM-1h-NLC surface connectivity efficiency. Connectivity efficiency equals connectivity to CTX- p The amount of CTX on the surface of -QM-1h-NLC divided by the amount of CTX at the theoretical sites in CTX-p-QM-1h-NLC.

[0040] For connection to CTX- p Protein integrity was tested on CTX on the surface of QM-1h-NLC. Primary structure analysis was performed using SDS-PAGE. After Coomassie Brilliant Blue staining, the bands of free CTX and CTX-linked proteins were compared. p Comparison of CTX banding on the QM-1h-NLC surface The tertiary structure of CTX was analyzed using a fluorescence spectrophotometer, and free CTX, thiolized CTX, and CTX- were compared. p The maximum emission wavelength of CTX attached to the surface of -QM-1h-NLC was determined, and the effects of the denaturant guanidine hydrochloride on the maximum emission wavelength and fluorescence intensity were also compared.

[0041] In addition to CTX-p In addition to performing nanoscale property detection using QM-1h-NLC, we also evaluated the CTX binding efficiency and antibody integrity. p -QM-1h-NLC was prepared using DSPE-PEG2000-Mal with a total lipid molar ratio of 0.1%, which was used to link CTX to construct CTX- p -QM-1h-NLC. Compared with other literature, the molar ratio of thiolated CTX to DSPE-PEG2000-Mal was reduced to 1.5:1; under these conditions, the CTX linkage efficiency (CE) reached 87.98±3.68%.

[0042] In addition, in order to determine the attachment on CTX- p To assess the antibody integrity of CTX on the surface of QM-1h-NLC, we determined the primary and tertiary structures of the protein. Figure 2 As shown in B, in addition, in order to determine the attachment on CTX- p To assess the antibody integrity of CTX on the surface of QM-1h-NLC, we determined the primary and tertiary structures of the protein. Figure 2 As shown in B, free CTX and CTX- p CTX on the surface of -QM-1h-NLC all showed two bands on the electrophoretic gel, namely heavy chain and light chain. This indicates that the CTX- p The CTX on the surface of the QM-1h-NLC maintains the integrity of the primary CTX structure and remains undamaged. For example... Figure 2 As shown in Figure C, under 280 nm excitation, due to the presence of aromatic amino acids in the protein structure, the maximum emission wavelength of free CTX is 346.67 ± 0.12 nm. Upon addition of guanidine hydrochloride (Gnd·HCl), the fluorescence spectrum of free CTX undergoes a spectral shift. max =355.67±0.31 nm), the fluorescence intensity decreased significantly. Thioylated CTX and coupled in CTX- p The maximum emission wavelengths of CTX on the surface of -QM-1h-NLC were 346.27±0.23 nm and 346.0±0.20 nm, respectively. After the addition of guanidine hydrochloride, the maximum emission wavelengths shifted to 356.2±0.60 nm and 356.2±0.42 nm, showing little change compared to the data from the free CTX sample group; this indicates that after two steps of thiolation and coupling, the coupling on the surface... p The tertiary structure of CTX on the surface of -QM-1h-NLC did not change significantly and did not affect the biological function of CTX.

[0043] Example 4: In vitro drug release The solution contains 0.5 mL of 2.48 mg / mL CTX- pThe dialysis bag for QM-1h-NLC solution (molecular weight cutoff 10000 Da) was placed in 50 mL of PBS (10 mM, pH 7.4) containing 1% Tween 20 and incubated at 37°C. o Place under C; and release. p -QM-1h48 hours. During the experiment, 0.5 mL of the release solution was taken at different time points, and 0.5 mL of fresh release solution was added. The concentration of [unspecified substance] in the release solution was determined using high-performance liquid chromatography (HPLC). p The concentration of -QM-1h was calculated. p -QM-1h cumulative release amount.

[0044] The sustained-release properties of nanomedicines are one of the core advantages that distinguishes nanocarriers from free drugs. Sustained release of nanomedicines can prolong the duration of drug action, maintain effective drug concentrations, and reduce the frequency of dosing. Therefore, we measured CTX- p -QM-1h-NLC in vitro drug release in PBS solution containing 1% Tween-20. (Example: ...) Figure 2 As shown in D, free p -QM-1h releases within the first hour and continues for approximately 8 hours; from the 8th hour onwards, drug release enters a plateau phase. CTX- p- QM-1h-NLC releases within 6 hours and continues for approximately 19 hours; from hour 24 onwards, drug release enters a plateau phase. CTX- p -QM-1h-NLC's drug release time is longer than that of free... p -QM-1h is 13 hours long, indicating that CTX- p -QM-1h-NLC can effectively prolong p -QM-1h release time.

[0045] Example 5 Antiproliferative activity 100 The L concentration is 1.0 x 10 4 Four T1 cells / mL were incubated in 96-well plates for 24 hours. Cells were then incubated with different concentrations of CTX-... p -QM-1h-NLC, CTX-NLC, Free p After incubating in fresh 1640 medium (QM-1h or CTX) for 48 hours, add 10 Prepare LMTT solution (5 mg / mL) and incubate in the dark for 4 hours. Record the absorbance at 570 nm using a microplate reader.

[0046] Table 54 Effects on cell viability in T1 cells ;

[0047] The 4T1 cell line is a typical cell line in breast cancer research, used to test CTX- p Effects of QM-1h-NLC on 4T1 cell proliferation. Table 5 shows the anti-proliferative activity of the drug on 4T1 cells, detected using the MTT assay. (Free...) p -QM-1h's antiproliferative activity is similar to that of CTX- p -QM-1h-NLC is similar, therefore free p -QM-1h load to CTX- p -QM-1h-NLC does not affect p -QM-1h's antiproliferative activity against 4T1 cells. CTX-NLC and CTX against... p -QM-1h did not contribute to the antiproliferative activity.

[0048] Example 6: In vivo anti-tumor assessment Balb / c mice (8 weeks old, female, 18-20g, n=35) were purchased from Pengyue Laboratory Animal Breeding Co., Ltd. and housed in the SPF animal facility. All animal studies were conducted in accordance with the U.S. National Laboratory Animal Care and Use Guidelines and were approved by the Ethics Committee of Liaocheng University (2022111013).

[0049] 0.15 mL of 4T1 cells at 1 x 10 7 The tumor was inoculated subcutaneously in the left axilla of Balb / c mice at a density of 1 cell / mL. On the third day, a noticeable tumor mass was palpable. The tumor-bearing mice were randomly divided into 6 groups of 5 mice each, and the drug was administered via tail vein injection daily. In addition, a group of 5 tumor-free normal mice was set up. (1) Normal group; (2) Model group; (3) CTX-NLC group; (4) 8 mg / kg p -QM-1h group (dissolved in physiological saline of 5% anhydrous ethanol and 5% polyoxyethylene (35) castor oil); (5) 2mg / kg CTX- p -QM-1h-NLC group; (6) 4mg / kgCTX- p -QM-1h-NLC group; (7) 8mg / kgCTX- p -QM-1h-NLC group. Injection volume of CTX-NLC group (80) L) and 8 mg / kg CTX- p -QM-1h-NLC group injection volume (80) L) equal, CTX-NLC group and 8mg / kg CTX- p The mice were compared with the QM-1h-NLC group. All mice were sacrificed on day 15, blood was collected from the eyes, and various organs were frozen in liquid nitrogen or immersed in buffered formalin (Beyotime).

[0050] To determine the in vivo antitumor activity, Balb / c mice carrying 4T1 tumors were established as a mouse tumor model. After subcutaneous injection of 4T1 cells, the tumor volume increased daily in the mice. Figure 3 As shown in A, 3B, and 3C, CTX- p -QM-1h-NLC effectively inhibited tumor growth in mice in a dose-dependent manner. 2 mg / kg CTX- p The inhibitory effect of QM-1h-NLC on tumors compared to 8 mg / kg of free drug p -QM-1h similarity. Unloaded CTX-NLC showed no significant tumor-suppressive effect, possibly due to the very low dose of CTX (1.55 mg / m²). 2 This is far below the recommended dose of CTX (initial dose 400 mg / m²). 2 Maintenance dose 50 mg / m 2 These data indicate that CTX- p -QM-1h-NLC can effectively inhibit tumor growth, while loading CTX-NLC onto... p -QM-1h can effectively enhance p -QM-1h anti-tumor effect.

[0051] Histopathological analysis of tumor tissue sections, such as Figure 3 As shown in Figure D, tumor sections in the model group show neatly arranged cells; while CTX- p Tumor sections from the QM-1h-NLC treatment group showed significant nuclear shrinkage. This indicates that CTX- p Intervention in QM-1h-NLC caused damage to tumor tissue.

[0052] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An immune nanostructured liposome with active EGFR targeting, characterized in that: Loading therapeutic active drugs p -QM-1h nanoliposomes are covalently coupled to cetuximab (CTX), which targets EGFR highly expressed in tumors; wherein the therapeutically active drug... p The structure of -QM-1h is shown below: ； The nanoliposomes comprise solid liposomes and liquid liposomes, wherein the solid liposomes are DSPC, Cholesterol, DSPE-PEG2000, and DSPE-PEG2000-MAL, and the liquid liposomes are one of Miglyol 812N, medium-chain triglyceride (MCT), castor oil, or oleic acid.

2. The immune nanostructured liposome according to claim 1, characterized in that: The molar ratio of DSPC, Cholesterol, DSPE-PEG2000 and DSPE-PEG2000-MAL is 1-4000:1-4000:1-600:1-30, preferably 3563:3563:490:

9.

3. The immune nanostructured liposome according to any one of claims 1-2, characterized in that: The molar ratio of solid liposomes to liquid liposomes is 2000-8000:2000-4000; preferably, the molar ratio is 7625:2375.

4. The immune nanostructured liposome according to any one of claims 1-2, characterized in that: The loaded therapeutic active drug p The nanoliposomes of -QM-1h are spherical with an average particle size ranging from 120nM to 180nM, preferably 150.53±0.68nM, and an encapsulation efficiency (EE) of 85% to 98%, preferably 87.98±3.68%.

5. The immune nanostructured liposome according to any one of claims 1-2, characterized in that: The thiolated CTX is covalently coupled to the maleimide site on the surface of the immune nanostructure liposome NLC; the thiolated CTX is generated by the reaction of the cetuximab CTX with Traut.

6. The immune nanostructured liposome according to any one of claims 1-2, wherein the molar ratio of cetuximab (CTX) to solid liposome DSPE-PEG2000-MAL is 1-15:1, preferably 1-10:1, more preferably 1-3:1, and most preferably 1.5:

1.

7. A method for preparing an immune nanostructured liposome with active EGFR targeting as described in any one of claims 1-6, comprising the following steps: (1) Loading therapeutically active drugs p Preparation of QM-1h nanoliposomes Solid liposomes DSPC, Cholesterol, DSPE-PEG2000, and DSPE-PEG2000-MAL were dissolved in an organic solvent; under heating conditions, the active substances were... p -QM-1h was dissolved in liquid liposomes; the solid liposome solution and the liquid liposome solution were mixed, and the organic solvent was removed by vacuum distillation; then PBS solution was added for hydration, the liposomes were ultrasonically broken, and the particle size was controlled by extrusion with a polycarbonate membrane to obtain the drug-loaded nanostructured lipid carrier. (2) Antibody-modified drug-loaded nanostructured liposomes CTX- p Preparation of QM-1h-NLC CTX and Traut reagent were mixed in PBS. The sulfurized CTX was dialyzed in PBS to remove excess Traut reagent. The sulfurized CTX was then mixed with the drug-loaded nanostructured lipids prepared in step (1). p Incubate with the QM-1h-NLC vector, then dialyze to remove unbound thiolated CTX to obtain the antibody-modified drug-loaded nanostructured liposomes.

8. The preparation method according to claim 7, characterized in that: In step (1), the organic solvent is selected from methanol, the PBS solution is 10 mM, pH 7.4, and the polycarbonate membrane is extruded through a 200 nM pore size. In step (2), the molar ratio of CTX to Traut reagent is 1:20, and the sulfurized CTX and p The molar ratio of -QM-1h-NLC is 1.5:

1.

9. The use of the immune nanostructured liposomes with active EGFR targeting as described in any one of claims 1-6 in the preparation of a medicament for treating tumor diseases.

10. The application according to claim 9, characterized in that: The tumor disease mentioned is breast cancer.

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