Nano-liposome delivery system targeting PLK1 / Aurora kinase and application of nano-liposome delivery system in oral squamous cell carcinoma treatment

Abstract

The invention belongs to the field of drug delivery, and discloses a nano-liposome delivery system targeting PLK1 / Aurora kinase and an application of the nano-liposome delivery system in oral squamous cell carcinoma treatment, the system jointly loads Volasertib and Alisertib in DOPE / CHEMS pH sensitive liposome, is coupled with GE11 targeting EGFR, realizes accurate delivery through active targeting and pH response drug release, induces mitosis disasters synergistically through double targets, and the inhibition rate exceeds 80%.

Description

Technical Field

[0001] This invention relates to the field of drug delivery system technology, specifically to a nanoliposome delivery system targeting PLK1 / Aurora kinase and its application in the treatment of oral squamous cell carcinoma. Background Technology

[0002] Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors of the head and neck, and its incidence is increasing year by year, accounting for more than 90% of all oral cancers. Although surgical resection combined with radiotherapy and chemotherapy has become the standard treatment for OSCC, the 5-year survival rate still hovers around 50%-60%, and the prognosis is even worse for patients with advanced stages. The highly invasive nature of OSCC, its high recurrence and metastasis rate, and its resistance to traditional chemotherapy drugs are the main reasons for treatment failure.

[0003] Mitotic catastrophe, a cell death mechanism triggered by abnormal mitosis, has been shown to play a crucial role in the development and progression of various malignant tumors. Polo-like kinase 1 (PLK1) and the Aurora kinase family are core kinases regulating mitotic processes, working synergistically in key events such as the G2 / M phase transition, spindle assembly, chromosome segregation, and cytokinesis. Studies have shown that PLK1 is activated by Aurora-A kinase through phosphorylation of its 210th threonine residue (Thr210), and a complex regulatory feedback loop exists between the two. Both PLK1 and Aurora-A are significantly overexpressed in various solid tumors, including OSCC, and are closely associated with tumor invasiveness, chemotherapy resistance, and poor prognosis.

[0004] Li et al. reported in Oncotarget (2015, 6(11): 9327-40) that the combination of the PLK1 inhibitor BI 2536 and an Aurora kinase inhibitor synergistically promotes mitotic catastrophe, especially showing significant selective killing effects on nasopharyngeal carcinoma cells. However, existing PLK1 inhibitors such as Volasetib (BI 6727) face many challenges in clinical trials, including dose-limiting toxicity due to myelosuppression, large fluctuations in blood drug concentrations, and limited efficacy in solid tumors. Similarly, although the Aurora-A kinase inhibitor Alisertib (MLN8237) has shown anti-tumor activity in various preclinical models, its poor water solubility, low bioavailability, and off-target effects limit its clinical application.

[0005] CN119367555A discloses a lipid nanoparticle drug delivery system, which co-loads metformin and... Nanoparticles enable synergistic therapy of tumor angiogenesis normalization and calcium overload. While this technology is innovative, it has the following limitations: First, the system only targets the improvement of the tumor microenvironment and lacks direct intervention on key targets of tumor cell mitosis; second, although anionic liposomes can encapsulate positively charged nanoparticles through electrostatic interactions, they lack active targeting capabilities, making it difficult to achieve precise drug delivery to specific tumor types; third, the system lacks an intelligent response drug release mechanism, making it impossible to achieve controlled drug release in the acidic tumor microenvironment.

[0006] Wang et al. reported in Science Advances (2024, 10(44): eadp7022) that they co-loaded Alisertib with the JAK2 inhibitor Ruxolitinib in nanocrystals to remodel the tumor immune microenvironment. However, this approach mainly focuses on the immunogenicity regulation of senescent cells rather than directly inducing mitotic catastrophe. In addition, the stability of the nanocrystal carrier and the controllability of drug release still need to be improved.

[0007] Epidermal growth factor receptor (EGFR) is universally overexpressed in OSCC, with overexpression or gene amplification occurring in approximately 70%-90% of OSCC cases. GE11 peptide (sequence YHWYGYTPQNVI) is a short peptide ligand that specifically recognizes EGFR, offering advantages over antibody ligands such as smaller molecular weight, lower immunogenicity, and ease of chemical modification. However, there is currently no technical approach combining GE11-targeted modification with a PLK1 / Aurora kinase dual-target inhibitor co-load strategy for OSCC treatment.

[0008] In summary, there is an urgent need in this field to develop a nanodelivery system that can simultaneously target PLK1 and Aurora kinase, possess active EGFR targeting capability, and achieve pH-responsive smart drug release, in order to overcome the shortcomings of existing technologies and provide a more effective treatment strategy for OSCC. Summary of the Invention

[0009] The purpose of this invention is to provide a nanoliposome delivery system targeting PLK1 / Aurora kinases to solve the problems of poor water solubility, low bioavailability, lack of active tumor targeting, and uncontrollable drug release in existing technologies for PLK1 and Aurora kinase inhibitors.

[0010] Another object of the present invention is to provide a method for preparing the above-described nanoliposome delivery system.

[0011] A first aspect of the present invention provides a nanoliposome delivery system targeting PLK1 / Aurora kinases, comprising a lipid nanoparticle carrier, a PLK1 inhibitor, an Aurora-A kinase inhibitor, and an EGFR targeting ligand; wherein the PLK1 inhibitor and the Aurora-A kinase inhibitor are co-loaded in a lipid bilayer and / or an aqueous core of the lipid nanoparticle carrier; the EGFR targeting ligand is linked to the surface of the lipid nanoparticle carrier via DSPE-PEG-Mal; and the lipid bilayer of the lipid nanoparticle carrier contains a pH-sensitive lipid component.

[0012] Further, the PLK1 inhibitor is selected from at least one of Volasetib, BI 2536, Rigosertib, and Onvansertib; preferably, the PLK1 inhibitor is Volasetib.

[0013] Further, the Aurora-A kinase inhibitor is selected from at least one of Alisertib, Danusertib, ENMD-2076, and MK-5108; preferably, the Aurora-A kinase inhibitor is Alisertib.

[0014] Furthermore, the EGFR targeting ligand is a GE11 polypeptide with the amino acid sequence YHWYGYTPQNVI.

[0015] Furthermore, the pH-sensitive lipid component comprises 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE) and cholesterol hemisuccinate (CHEMS); the molar ratio of DOPE to CHEMS is 2:1 to 4:1.

[0016] Furthermore, the lipid bilayer of the lipid nanoparticle carrier further includes 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and DSPE. -Mal; the DSPC, DOPE, CHEMS, cholesterol and DSPE- The molar ratio of -Mal is 20-30:30-40:10-15:35-45:2-5.

[0017] Further, the molar ratio of the PLK1 inhibitor to the Aurora-A kinase inhibitor is 1:1 to 1:3; preferably, the molar ratio is 1:2.

[0018] Furthermore, the average particle size of the nanoliposome delivery system is 80-150 nm; preferably, the average particle size is 100-120 nm.

[0019] Furthermore, the zeta potential of the nanoliposome delivery system is -15mV to -5mV.

[0020] Furthermore, the cumulative release rate of the nanoliposome delivery system is less than 20% in 24 hours at pH 7.4, 40%-60% in 24 hours at pH 6.5, and more than 80% in 24 hours at pH 5.5.

[0021] A second aspect of the present invention provides a method for preparing the above-mentioned nanoliposome delivery system, comprising the following steps: dissolving a lipid component in an organic solvent, adding a PLK1 inhibitor and an Aurora-A kinase inhibitor, removing the organic solvent by rotary evaporation under reduced pressure to form a lipid film; hydrating the lipid film with an aqueous medium, sonicating it, and then extruding it through an extruder to prepare liposomes; coupling an EGFR targeting ligand to the surface of the liposomes via a thiol-maleimide reaction to obtain the nanoliposome delivery system.

[0022] Furthermore, the lipid components include DSPC, DOPE, CHEMS, cholesterol, and DSPE- -Mal.

[0023] Furthermore, the organic solvent is selected from chloroform, anhydrous ethanol, or mixtures thereof.

[0024] Furthermore, the hydration temperature is 45-55℃, and the hydration time is 30-60 min.

[0025] Furthermore, the filter membrane pore sizes of the extruder are 400nm, 200nm and 100nm respectively, and the extrusion times for each pore size are 10-20 times.

[0026] Furthermore, the EGFR targeting ligand is modified with a terminal cysteine ​​residue before coupling, and the coupling reaction is carried out in a buffer solution at pH 6.5-7.5 for 2-4 hours.

[0027] The nanoliposome delivery system of the present invention has the following beneficial effects:

[0028] First, this invention innovatively co-loads the PLK1 inhibitor Volasetib and the Aurora-A kinase inhibitor Alisertib into the same nanoliposome, achieving synergistic dual-target intervention on the mitotic regulatory network. The PLK1 inhibitor can block the G2 / M phase transition and spindle assembly, while the Aurora-A inhibitor can prevent centrosome maturation and the establishment of mitotic spindle polarity. The combination of the two can produce a synergistic mitotic catastrophe-inducing effect, significantly enhancing anti-tumor activity.

[0029] Second, this invention endows the drug delivery system with intelligent responsive drug release capabilities by introducing pH-sensitive components such as DOPE and CHEMS into the lipid bilayer. The carboxyl groups of CHEMS are deprotonated at neutral pH, forming hydrogen bonds with the amide groups of DOPE to maintain the layered phase structure of the lipid bilayer. When entering the acidic tumor microenvironment (pH 6.5-6.8) or lysosomes (pH 4.5-5.5), the carboxyl groups of CHEMS protonate, leading to hydrogen bond breakage. The lipid membrane transitions from a layered phase to a hexagonal phase, triggering membrane fusion and rapid drug release, thereby achieving spatiotemporally controllable and precise drug delivery.

[0030] Third, this invention covalently links the GE11 peptide to the liposome surface via DSPE-PEG-Mal, achieving active targeted recognition of OSCC cells highly expressing EGFR. The dissociation constant of the GE11 peptide and EGFR is also discussed. With a molecular weight of approximately 22 nM, it can effectively mediate receptor-mediated endocytosis of nanoliposomes, significantly increasing drug accumulation at tumor sites while reducing non-specific distribution to normal tissues.

[0031] Fourth, the nanoliposome delivery system of the present invention has a uniform particle size (100-120nm), good blood circulation stability and EPR effect, and can achieve tumor enrichment through a dual mechanism of passive and active targeting. It is expected to increase the tumor inhibition rate to more than 80% while reducing the system toxicity by more than 50%. Attached Figure Description

[0032] Figure 1 This is a particle size distribution diagram of the nanoliposomes prepared in Example 1 of the present invention.

[0033] Figure 2 The cumulative release curves of the nanoliposomes prepared in Example 1 of this invention under different pH conditions are shown.

[0034] Figure 3 This is a graph showing the cell viability test results of Cal-27 cells after different treatments in the test examples of this invention.

[0035] Figure 4 This is a cell cycle distribution diagram of Cal-27 cells after different treatments in the test examples of this invention.

[0036] Figure 5 This is a tumor growth curve of the xenograft tumor model in the test examples of this invention. Detailed Implementation

[0037] Please refer to the attached document. Figures 1-5The technical solutions of the present invention will be clearly and completely described below with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, can be obtained commercially.

[0038] Example 1: Preparation of a nanoliposome delivery system targeting PLK1 / Aurora kinase

[0039] This embodiment describes the preparation of a nanoliposome delivery system targeting PLK1 / Aurora kinase. The specific steps are as follows:

[0040] Step 1: Preparation of lipid films

[0041] Accurately weigh 15.8 mg of DSPC, 18.6 mg of DOPE, 4.8 mg of CHEMS, 7.7 mg of cholesterol, and DSPE- -Mal 2.9mg was prepared, with a molar ratio of 25:35:12:40:2. The lipid fraction was placed in a 100mL round-bottom flask, and 10mL of a mixture of chloroform and anhydrous ethanol (volume ratio 3:1) was added. The mixture was then sonicated until completely dissolved. Volasetib 2.5mg and Alisertib 2.2mg (molar ratio approximately 1:2) were weighed out and added to the lipid solution, and mixed thoroughly. The round-bottom flask was placed on a rotary evaporator with a water bath temperature of 40℃ and a rotation speed of 60rpm. The organic solvent was removed by rotary evaporation under reduced pressure, forming a uniform and transparent lipid film on the flask wall. The round-bottom flask was then placed in a vacuum drying oven and evacuated at room temperature for 2 hours to completely remove residual solvent.

[0042] Step 2: Preparation of liposomes

[0043] Add 5 mL of PBS buffer (pH 7.4) to a flask containing the lipid film and hydrate in a 50°C water bath for 45 min, vortexing for 1 min every 10 min to promote complete hydration and detachment of the lipid film. Transfer the hydrated suspension to a probe sonicator and sonicate at 200 W for 5 min (on for 2 s, off for 3 s) under ice bath conditions to obtain primary liposomes. Extrude the primary liposomes sequentially through 400 nm, 200 nm, and 100 nm polycarbonate membranes 15 times each to obtain liposomes with uniform particle size.

[0044] Step 3: Conjugation of GE11 peptide

[0045] A terminal cysteine-modified GE11 peptide (Cys-YHWYGYTPQNVI) with a purity greater than 95% was synthesized. 8 mg of the modified GE11 peptide was dissolved in 2 mL of pH 7.0 PBS buffer and slowly added dropwise to the aforementioned liposome solution. The mixture was incubated at room temperature with stirring for 3 h to allow the thiol groups to undergo a Michael addition reaction with the maleimide groups. After the reaction, unreacted free peptides were removed by dialysis (molecular weight cutoff 100 kDa). The dialysate was pH 7.4 PBS buffer, and dialysis was performed for 24 h with the dialysate changed three times. After dialysis, the solution was concentrated to the desired concentration using ultrafiltration to obtain the nanoliposome delivery system targeting PLK1 / Aurora kinase (denoted as GE11-pH-LNP / V+A).

[0046] Dynamic light scattering (DLS) analysis revealed that the average particle size of the prepared GE11-pH-LNP / V+A was 112.5 ± 3.8 nm, the polydispersity index (PDI) was 0.156 ± 0.021, and the zeta potential was -10.3 ± 1.2 mV. HPLC analysis determined the encapsulation efficiency: 87.2 ± 2.1% for Volasertib and 91.5 ± 1.8% for Alisertib.

[0047] Example 2 Preparation of non-targeted pH-sensitive liposomes

[0048] In this embodiment, pH-sensitive liposomes without the GE11 targeting ligand were prepared as a control. The specific steps are as follows:

[0049] Liposomes were prepared according to steps 1 and 2 of Example 1, wherein the lipid component contained DSPE- -Mal is replaced with an equimolar amount of DSPE- The remaining steps are the same as in Example 1. The resulting non-targeted pH-sensitive liposomes are denoted as pH-LNP / V+A.

[0050] DLS analysis showed that the average particle size of pH-LNP / V+A was 108.3±4.2 nm, the PDI was 0.142±0.018, and the Zeta potential was -12.8±1.5 mV. The encapsulation efficiency of Volasertib was 89.1±1.9%, and that of Alisertib was 92.8±2.3%.

[0051] Example 3 Preparation of non-pH-sensitive targeting liposomes

[0052] In this embodiment, targeted liposomes without pH-sensitive components were prepared as a control. The specific steps are as follows:

[0053] Following the method of Example 1, DOPE and CHEMS in the lipid component were replaced with equimolar amounts of DSPC and lecithin, with the remaining steps being the same as in Example 1. The resulting non-pH-sensitive targeting liposome was designated GE11-LNP / V+A.

[0054] DLS analysis showed that the average particle size of GE11-LNP / V+A was 115.8±5.1 nm, the PDI was 0.168±0.025, and the Zeta potential was -8.7±1.8 mV. The encapsulation efficiency of Volasertib was 85.6±2.5%, and that of Alisertib was 88.9±2.7%.

[0055] Example 4 Preparation of single-drug-loaded liposomes

[0056] In this embodiment, liposomes loaded with only Volasertib or Alisertib were prepared as controls.

[0057] Example 4-1: Preparation of GE11-pH-LNP / V

[0058] Following the method of Example 1, only 2.5 mg of Volasetib was added in the lipid film preparation step, without Alisertib, and the remaining steps were the same as in Example 1.

[0059] Example 4-2: Preparation of GE11-pH-LNP / A

[0060] Following the method of Example 1, only 2.2 mg of Alisertib was added in the lipid film preparation step, without adding Volasertib, and the remaining steps were the same as in Example 1.

[0061] Example 5: Optimization of Lipid Occurrence Ratio

[0062] This example examines the effect of the DOPE to CHEMS molar ratio on pH responsiveness.

[0063] A series of pH-sensitive liposomes were prepared according to the lipid group ratios shown in Table 1, and their drug release behavior under different pH conditions was measured.

[0064] Table 1 Optimization of lipid group allocation

[0065] Formula number DSPC (Mollions) DOPE (molar fractions) CHEMS (moles) Cholesterol (molarities) F1 25 20 20 40 F2 25 30 15 40 F3 25 35 12 40 F4 25 40 10 40 F5 25 45 8 40

[0066] Drug release experiments showed that when the DOPE / CHEMS molar ratio was 35:12 (approximately 3:1), the liposomes exhibited the best stability at pH 7.4 (24h release rate <15%), while the release rate was fastest at pH 5.5 (24h release rate >85%), demonstrating the most significant pH responsiveness. When the DOPE ratio was too low, the pH sensitivity of the liposomes decreased; when the DOPE ratio was too high, the stability of the liposomes under neutral conditions decreased.

[0067] Example 6: Optimization of Drug Ratio

[0068] This embodiment examines the effect of the molar ratio of Volasertib to Alisertib on antitumor activity.

[0069] A series of GE11-pH-LNP / V+A drugs were prepared according to the drug molar ratios shown in Table 2, and their in vitro inhibitory activity against Cal-27 oral squamous cell carcinoma cells was determined by the MTT assay.

[0070] Table 2 Optimization of Drug Ratios

[0071] Formula number Volasertib:Alisertib (molar ratio) (nM) D1 1:0.5 185.3±12.6 D2 1:1 142.8±8.9 D3 1:2 98.5±6.3 D4 1:3 102.1±7.5 D5 1:4 118.9±9.2

[0072] The results showed that the formulation exhibited the strongest inhibitory activity against Cal-27 cells when the molar ratio of Volasetib to Alisertib was 1:2. The value was the lowest. This may be related to the relative contributions of PLK1 and Aurora-A in mitosis regulation and their synergistic pharmacodynamic mechanism.

[0073] Example 7 In vitro drug release experiment

[0074] This embodiment measures the drug release behavior of GE11-pH-LNP / V+A under different pH conditions.

[0075] 1 mL of GE11-pH-LNP / V+A (drug concentrations of Volasertib 0.5 mg / mL and Alisertib 0.44 mg / mL) was placed in a dialysis bag (molecular weight cutoff 10 kDa) and then placed in 50 mL of PBS buffer at pH 7.4, pH 6.5, and pH 5.5, respectively, and shaken at 37°C and 100 rpm. At predetermined time points (0.5, 1, 2, 4, 6, 8, 12, and 24 h), 1 mL of the dialysis fluid was collected and an equal volume of fresh buffer was added. The concentrations of Volasertib and Alisertib in the dialysis fluid were determined by HPLC, and the cumulative release percentage was calculated.

[0076] The results showed that at pH 7.4, the 24-hour cumulative release rates of Volasertib and Alisertib in GE11-pH-LNP / V+A were 12.3±1.8% and 14.6±2.1%, respectively, indicating that the liposomes had good stability in a simulated blood circulation environment. At pH 6.5 (simulating the tumor microenvironment), the 24-hour cumulative release rates increased to 48.5±3.7% and 52.8±4.2%, respectively. At pH 5.5 (simulating the lysosomal environment), the drug release rate was significantly accelerated, with 24-hour cumulative release rates reaching 86.2±4.5% and 89.7±3.9%, respectively. These results confirm that GE11-pH-LNP / V+A has good pH-responsive drug release characteristics.

[0077] As a control, the cumulative release rates of the non-pH-sensitive liposome GE11-LNP / V+A at pH 7.4, 6.5 and 5.5 did not differ significantly over 24 hours, all falling within the range of 20%-30%, confirming the necessity of pH-sensitive lipid components DOPE and CHEMS for achieving intelligent responsive drug release.

[0078] Test Example 1: In Vitro Cell Take-Up Experiment

[0079] This test case examines the effect of GE11 targeting ligands on liposome cell uptake efficiency.

[0080] Liposomes were labeled with coumarin-6 to prepare GE11-pH-LNP / Cou-6 and pH-LNP / Cou-6. Cal-27 cells were seeded in 6-well plates. Cells were cultured for 24 hours, and then cultured in medium containing GE11-pH-LNP / Cou-6 or pH-LNP / Cou-6 (Coumarin-6 final concentration 100 ng / mL) for 1, 2, and 4 hours respectively. Cells were collected and intracellular fluorescence intensity was detected by flow cytometry (BD FACSCalibur).

[0081] The results showed that, under the same incubation time, the cellular fluorescence intensity of the GE11-pH-LNP / Cou-6 group was significantly higher than that of the pH-LNP / Cou-6 group. After 4 hours of incubation, the mean fluorescence intensity (MFI) of the GE11-pH-LNP / Cou-6 group was 3.2 times that of the pH-LNP / Cou-6 group (p<0.001). To verify that this enhanced uptake effect depends on EGFR-mediated receptor endocytosis, cells were pretreated with free GE11 peptide (50 μg / mL) for 1 hour for a competitive inhibition experiment. The results showed that GE11 pretreatment reduced the uptake efficiency of GE11-pH-LNP / Cou-6 by 65% ​​(p<0.001), confirming that the GE11-targeting ligand can significantly enhance cellular uptake of liposomes through EGFR-mediated mechanisms.

[0082] Test Example 2: In vitro cytotoxicity experiment

[0083] This test case investigated the in vitro inhibitory activity of GE11-pH-LNP / V+A against oral squamous cell carcinoma cell lines.

[0084] Two OSCC cell lines were selected: Cal-27 (high EGFR expression) and SCC-25 (moderate EGFR expression). Cells were seeded in 96-well plates. Cells were cultured (cells / well) for 24 hours, and then cultured for another 48 hours in media containing different concentrations of free drug combinations (Free V+A), pH-LNP / V+A, GE11-LNP / V+A, or GE11-pH-LNP / V+A. Cell viability was determined using the MTT assay. value.

[0085] The results showed that for Cal-27 cells, GE11-pH-LNP / V+A The value was 98.5±6.3 nM, significantly lower than GE11-LNP / V+A (168.2±11.5 nM), pH-LNP / V+A (245.6±18.3 nM), and Free V+A (312.4±25.7 nM) (p<0.001). For SCC-25 cells, GE11-pH-LNP / V+A also exhibited the strongest inhibitory activity. The value was 135.8 ± 9.7 nM. These results indicate that GE11 targeting modification and pH-sensitive design synergistically enhance the antitumor activity of nanoliposomes, with a more significant effect on Cal-27 cells that highly express EGFR.

[0086] To verify the synergistic effect of dual-target inhibition of PLK1 and Aurora-A, the Chou-Talalay co-inhibition index (CI) method was used to analyze the interaction between Volasertib and Alisertib. The results showed that treatment of Cal-27 cells with GE11-pH-LNP / V+A resulted in... , and The CI values ​​at the different concentrations were 0.52, 0.48, and 0.45, all less than 1, indicating that the two drugs have a significant synergistic effect.

[0087] Test Example 3: Cell Cycle and Apoptosis Analysis

[0088] This test case investigated the effects of GE11-pH-LNP / V+A on cell cycle distribution and apoptosis in Cal-27 cells.

[0089] Cal-27 cells were seeded in 6-well plates. After culturing for 24 hours, PBS (control group), Free V+A, pH-LNP / V+A, GE11-LNP / V+A, or GE11-pH-LNP / V+A (drug concentrations of 100 nM for Volasetib and 200 nM for Alisertib) were added to each well, and the mixture was cultured for another 24 hours.

[0090] For cell cycle analysis, cells were collected, fixed overnight with 70% ethanol, and incubated in PBS solution containing RNase A (100 μg / mL) and propidium iodide (50 μg / mL) at 37°C in the dark for 30 min. Flow cytometry analysis was then performed. Results showed that the proportion of cells in the G2 / M phase was 18.5±2.1% in the control group; 38.6±3.2% in the Free V+A group; 45.2±3.8% in the pH-LNP / V+A group; 52.8±4.1% in the GE11-LNP / V+A group; and 68.5±4.7% in the GE11-pH-LNP / V+A group. The GE11-pH-LNP / V+A group exhibited the most significant G2 / M phase arrest, and the proportion of cells in the Sub-G1 phase (apoptotic cells) reached 25.3±2.8%, significantly higher than the other groups.

[0091] For apoptosis analysis, Annexin V-FITC / PI double staining was used. Cells were collected, resuspended in binding buffer, and incubated with Annexin V-FITC (5 μL) and PI (5 μL) for 15 min at room temperature in the dark. Flow cytometry was then used for analysis. The results showed that the total apoptosis rate (early + late) in the GE11-pH-LNP / V+A group was 52.6±4.3%, significantly higher than that in the GE11-LNP / V+A group (38.5±3.6%), pH-LNP / V+A group (28.7±2.9%), Free V+A group (22.4±2.5%), and control group (5.2±0.8%) (p<0.001).

[0092] Test Example 4: Mitotic Catastrophe Detection

[0093] This test case examines the ability of GE11-pH-LNP / V+A to induce mitotic catastrophe in Cal-27 cells.

[0094] Cal-27 cells were seeded in confocal culture dishes and treated as in Test Example 3. After 24 h of treatment, the cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 for 10 min, and blocked with 5% BSA for 1 h. Anti-α-Tubulin antibody (1:200) was added and incubated overnight at 4°C. After washing with PBS, Alexa Fluor 488-labeled secondary antibody (1:500) was added and incubated at room temperature for 1 h. The cells were mounted with DAPI-containing mounting medium, and the spindle and nuclear morphology were observed using a laser confocal microscope (Zeiss LSM 880).

[0095] The results showed that the control group cells exhibited a normal bipolar spindle structure; while the GE11-pH-LNP / V+A treatment group showed obvious mitotic catastrophe characteristics, including multipolar spindles (accounting for 45.2% of mitotic cells), spindle breakage (32.8%), chromosome misalignment (58.6%), and multinucleated giant cell formation (15.3%). The proportion of these abnormal mitotic phenotypes was significantly higher than that of other treatment groups (p<0.001), confirming that dual inhibition of PLK1 and Aurora-A can effectively induce mitotic catastrophe.

[0096] Test Example 5: Western Blot Detection

[0097] This test case uses Western blotting to detect the effect of GE11-pH-LNP / V+A on the expression of mitosis-related proteins.

[0098] Cal-27 cells were treated as described in Test Example 3 for 24 hours, and total protein was extracted. Protein concentration was determined using the BCA method. An equal amount of protein (30 μg) was subjected to SDS-PAGE electrophoresis, transferred to a PVDF membrane, and blocked with 5% skim milk powder for 1 hour. Antibodies against PLK1 (1:1000), p-PLK1-Thr210 (1:1000), Aurora-A (1:1000), p-Aurora-A-Thr288 (1:1000), Cyclin B1 (1:1000), p-Histone H3-Ser10 (1:1000), cleaved PARP (1:1000), cleaved Caspase-3 (1:1000), and GAPDH (1:5000) were added, and the cells were incubated overnight at 4°C. After washing with TBST, add HRP-labeled secondary antibody (1:5000), incubate at room temperature for 1 h, and then perform ECL luminescence development.

[0099] The results showed that GE11-pH-LNP / V+A treatment significantly reduced the expression levels of p-PLK1-Thr210 and p-Aurora-A-Thr288, by 78.5% and 82.3%, respectively, confirming that the drug effectively inhibited the kinase activity of PLK1 and Aurora-A. Simultaneously, Cyclin B1 expression increased 3.2-fold, and p-Histone H3-Ser10 expression increased 2.8-fold, indicating cell arrest in the M phase. Cleaved PARP and cleaved Caspase-3 expression levels increased 4.5-fold and 3.8-fold, respectively, confirming that the drug induced Caspase-dependent apoptosis.

[0100] Test Example 6: In vivo pharmacokinetic experiment

[0101] This test case investigates the pharmacokinetic characteristics of GE11-pH-LNP / V+A in BALB / c mice.

[0102] BALB / c mice (female, 6-8 weeks old, weighing 18-22 g) were randomly divided into two groups (n=6), receiving either Free V+A or GE11-pH-LNP / V+A via tail vein injection (both doses: Volasertib 5 mg / kg, Alisertib 8.8 mg / kg). Blood samples of 0.3 mL were collected from the orbital sinus at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-administration. Plasma was separated, and the plasma concentrations of Volasertib and Alisertib were determined by HPLC-MS / MS. Pharmacokinetic parameters were fitted using DAS 2.0 software.

[0103] The results showed that the elimination half-life of Volasetib in GE11-pH-LNP / V+A was ( The mean 0.001 h was 8.5 ± 1.2 h, significantly longer than the 2.1 ± 0.3 h in the Free V+A group (p < 0.001); the area under the curve (AUC) was 8.5 ± 1.2 h. The level of Alisertib was 4.8 times that of the Free V+A group. The duration of the illness increased from 1.8 ± 0.2 h to 7.2 ± 0.9 h. It was 5.2 times that of the Free V+A group. These results indicate that nanoliposome delivery systems can significantly prolong drug circulation time in vivo and improve drug bioavailability.

[0104] Test Example 7: In vivo antitumor activity experiment

[0105] This test case investigated the in vivo antitumor activity of GE11-pH-LNP / V+A against a Cal-27 xenograft tumor model.

[0106] BALB / c nude mice (female, 4-6 weeks old) were subcutaneously injected with Cal-27 cell suspension in the right axilla. 100 μL per cell / animal. When the tumor volume reaches 80-120... At that time, patients were randomly divided into 6 groups (n=8): saline control group, Free V+A group, GE11-pH-LNP / V group, GE11-pH-LNP / A group, pH-LNP / V+A group, and GE11-pH-LNP / V+A group. Volasertib was administered via tail vein on days 0, 3, 6, and 9, with doses of 5 mg / kg (relevant group) and / or 8.8 mg / kg (relevant group) in each group. Tumor volume and body weight were measured every 2 days. Tumor volume was calculated using the formula: Where L is the long diameter of the tumor and W is the short diameter. Mice were sacrificed on day 21, and tumors were collected, weighed, and the tumor inhibition rate was calculated.

[0107] The results showed that the tumor volume growth was the slowest in the GE11-pH-LNP / V+A group, with a mean tumor volume of 158.3 ± 35.6 mm on day 21. The value was significantly lower than that of the control group (985.2±142.5). ), Free V+A group (612.8±98.3 ), GE11-pH-LNP / V group (425.6±72.8) ), GE11-pH-LNP / A group (398.5±68.2) () and pH-LNP / V+A group (285.3±52.6) (p<0.001). Regarding tumor inhibition rate, the GE11-pH-LNP / V+A group achieved 83.9%, while the Free V+A group only achieved 37.8%, the pH-LNP / V+A group achieved 71.0%, the GE11-pH-LNP / V group achieved 56.8%, and the GE11-pH-LNP / A group achieved 59.6%.

[0108] Regarding changes in body weight among the different groups of mice, the Free V+A group showed a significant decrease in body weight (12.5±2.3%), while the GE11-pH-LNP / V+A group showed a smaller change in body weight (3.2±0.8%), indicating that the nanoliposome delivery system can significantly reduce the systemic toxicity of the drug.

[0109] Test Example 8: Immunohistochemical Analysis of Tumor Tissue

[0110] This test case involves immunohistochemical analysis of the tumor tissue collected in Test Case 7.

[0111] Tumor tissues from each group were collected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (4μm). Immunohistochemical staining was performed for Ki-67 (proliferation marker), p-Histone H3-Ser10 (mitotic marker), and cleaved Caspase-3 (apoptosis marker).

[0112] The results showed that the proportion of Ki-67 positive cells in the tumor tissue of the GE11-pH-LNP / V+A group was 12.3±2.5%, which was significantly lower than that of the control group (68.5±5.8%) (p<0.001); the proportion of p-Histone H3-Ser10 positive cells was 35.2±4.6%, which was significantly higher than that of the control group (8.6±1.2%), indicating that the tumor cells were arrested in the mitotic phase; the proportion of cleaved Caspase-3 positive cells was 42.8±5.3%, which was significantly higher than that of the control group (3.2±0.6%), confirming that the drug induced tumor cell apoptosis.

[0113] Comparative Example 1: Liposomes without pH-sensitive components

[0114] GE11-LNP / V+A was prepared according to the method in Example 3, and its in vivo antitumor activity was investigated.

[0115] Using the same animal model and dosing regimen as in Test Case 7, the mean tumor volume in the GE11-LNP / V+A group was 425.3 ± 68.5 mm on day 21. The tumor inhibition rate was 56.8%, significantly lower than the 83.9% in the GE11-pH-LNP / V+A group (p<0.01). These results indicate that pH-sensitive design is crucial for achieving intelligent drug release and enhancing antitumor activity.

[0116] Comparative Example 2: Liposomes without GE11 targeting ligand

[0117] pH-LNP / V+A was prepared according to the method in Example 2, and its in vivo antitumor activity was investigated.

[0118] Using the same animal model and drug administration regimen as in Test Case 7, the mean tumor volume in the pH-LNP / V+A group was 285.3 ± 52.6 mm on day 21. The tumor inhibition rate was 71.0%, lower than the 83.9% in the GE11-pH-LNP / V+A group (p<0.05). These results indicate that GE11-targeted modification can further enhance the antitumor activity of nanoliposomes through an active targeting mechanism.

[0119] Comparative Example 3: Single-drug liposomes

[0120] GE11-pH-LNP / V and GE11-pH-LNP / A were prepared according to the methods in Examples 4-1 and 4-2, respectively, to investigate the difference in antitumor activity between single-drug liposomes and dual-drug liposomes.

[0121] Using the same animal model and dosing regimen as in Test Case 7, the mean tumor volume in the GE11-pH-LNP / V group was 425.6 ± 72.8 mm on day 21. The tumor inhibition rate was 56.8%; the mean tumor volume in the GE11-pH-LNP / A group was 398.5 ± 68.2 mm. The tumor inhibition rate was 59.6%. The tumor inhibition rate of the GE11-pH-LNP / V+A group was 83.9%, which was significantly higher than the expected value of the sum of the two single-drug groups (about 85%), indicating that the dual-target inhibition of PLK1 and Aurora-A has a synergistic effect.

[0122] The GE11-pH-LNP / V+A nanoliposome delivery system of this invention achieves highly efficient antitumor effects through multiple synergistic mechanisms. First, the GE11 peptide-targeting ligand specifically recognizes EGFR, which is highly expressed on the surface of OSCC cells, enabling efficient cellular uptake of the nanoliposomes via receptor-mediated endocytosis. Second, the pH-sensitive lipid components DOPE and CHEMS trigger a lipid membrane phase transition in the acidic environment of the lysosome, achieving rapid drug release. The released Volasertib inhibits PLK1 kinase activity, blocking the G2 / M phase transition and spindle assembly; simultaneously, Alisertib inhibits Aurora-A kinase activity, preventing centrosome maturation and mitotic entry. The dual inhibition of PLK1 and Aurora-A produces a synergistic effect, inducing severe mitotic catastrophe in tumor cells, manifested as multipolar spindle formation, chromosome segregation disorder, and the formation of multinucleated giant cells, ultimately leading to cell death via the caspase-dependent apoptosis pathway. In vivo experiments have demonstrated that GE11-pH-LNP / V+A can achieve a tumor inhibition rate of 83.9% while significantly reducing systemic toxicity, providing a new strategy for the clinical treatment of OSCC.

[0123] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A nanoliposome delivery system targeting PLK1 / Aurora kinase, characterized in that: The invention comprises a lipid nanoparticle carrier, a PLK1 inhibitor, an Aurora-A kinase inhibitor, and an EGFR targeting ligand; the PLK1 inhibitor and the Aurora-A kinase inhibitor are co-loaded in the lipid bilayer and / or aqueous core of the lipid nanoparticle carrier; the EGFR targeting ligand is linked to the surface of the lipid nanoparticle carrier via DSPE-PEG-Mal; and the lipid bilayer of the lipid nanoparticle carrier contains a pH-sensitive lipid component.

2. The nanoliposome delivery system according to claim 1, characterized in that: The PLK1 inhibitor is selected from at least one of Volasertib, BI 2536, Rigosertib, and Onvansertib.

3. The nanoliposome delivery system according to claim 1, characterized in that: The Aurora-A kinase inhibitor is selected from at least one of Alisertib, Danusertib, ENMD-2076, and MK-5108.

4. The nanoliposome delivery system according to claim 1, characterized in that: The EGFR targeting ligand is the GE11 polypeptide, whose amino acid sequence is YHWYGYTPQNVI.

5. The nanoliposome delivery system according to claim 1, characterized in that: The pH-sensitive lipid component comprises 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine and cholesterol hemisuccinate; the molar ratio of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine and cholesterol hemisuccinate is 2:1 to 4:

1.

6. The nanoliposome delivery system according to claim 5, characterized in that: The lipid bilayer of the lipid nanoparticle carrier further includes 1,2-distearate-sn-glycerol-3-phosphocholine, cholesterol, and DSPE. -Mal; the 1,2-distearate-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, cholesterol hemisuccinate, cholesterol and DSPE- The molar ratio of -Mal is 20-30:30-40:10-15:35-45:2-5.

7. The nanoliposome delivery system according to any one of claims 1 to 6, characterized in that: The molar ratio of the PLK1 inhibitor to the Aurora-A kinase inhibitor is 1:1 to 1:

3.

8. The nanoliposome delivery system according to any one of claims 1 to 6, characterized in that: The average particle size of the nanoliposome delivery system is 80-150 nm; and / or, the zeta potential of the nanoliposome delivery system is -15 mV to -5 mV.

9. The nanoliposome delivery system according to any one of claims 1 to 6, characterized in that: The preparation method includes the following steps: dissolving the lipid component in an organic solvent, adding a PLK1 inhibitor and an Aurora-A kinase inhibitor, removing the organic solvent by rotary evaporation under reduced pressure to form a lipid film; adding an aqueous medium to hydrate the lipid film, sonicating it, and then extruding it through an extruder to prepare liposomes; coupling an EGFR targeting ligand to the surface of the liposomes via a thiol-maleimide reaction to obtain the final product.

10. The use of the nanoliposome delivery system of claim 9 in the preparation of a drug for treating oral squamous cell carcinoma.

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