A biomimetic hybrid liposome co-loading senolytic drugs and antibiotics, and a preparation method and application thereof

Abstract

This invention discloses a biomimetic hybrid liposome nanomedicine co-loaded with senolytic drugs and antibiotics, its preparation method, and its applications. The nanomedicine uses egg yolk lecithin and cholesterol as lipid materials. The Bcl-2 / Bcl-xL inhibitor ABT-263 is loaded into a lipid bilayer, and the antibiotic ciprofloxacin hydrochloride is encapsulated in an aqueous core. After preparing co-loaded liposomes using a thin-film hydration-ultrasound-extrusion-ultrafiltration method, the liposomes are fused with doxorubicin-induced senescent tumor cell membranes via ultrasound-assisted physical extrusion. The resulting biomimetic hybrid liposome possesses multiple functions, including homologous targeting of senescent tumor cells, selective removal of senescent cells, and killing of senescent bacteria. Combined use with 5-fluorouracil can enhance the killing effect on senescent tumor cells. This invention belongs to the field of biomedical nanotechnology.

Description

Technical Field

[0001] This invention belongs to the field of biomedical nanotechnology, specifically relating to a biomimetic hybrid liposome nanomedicine based on the membrane of senescent tumor cells and its preparation method, as well as the application of this nanomedicine in the preparation of targeted removal of senescent tumor cells and antibacterial drugs. Background Technology

[0002] Colorectal cancer is a malignant tumor with the highest incidence and mortality rates worldwide. Chemotherapy based on 5-fluorouracil (5-FU) is one of its main treatment methods, but the occurrence of chemotherapy resistance seriously limits the further improvement of clinical efficacy.

[0003] Recent studies have shown that *E. coli* pks⁺ in the gut microenvironment can synthesize and release the genotoxin colibactin through the non-ribosomal peptide / polyketide synthase system encoded by the pks gene island in its genome. This toxin can directly induce interstrand cross-linking and double-strand breaks in host cell DNA, leading to irreversible cellular senescence in tumor cells. Although senescent tumor cells lose their proliferative capacity, they are not in a "quiet and harmless" state. They remodel the tumor microenvironment by secreting large amounts of senescence-associated secretory phenotype (SASP) factors (such as IL-6, IL-8, and MMP-3), promoting angiogenesis, immune escape, and invasion and metastasis of neighboring tumor cells. More importantly, senescent tumor cells exhibit significant resistance to chemotherapy drugs, becoming a potential driver of chemotherapy failure and tumor recurrence.

[0004] Senolytic strategies are a therapeutic approach proposed in recent years for the selective elimination of senescent cells. ABT-263 (Navitoclax), as a Bcl-2 / Bcl-xL protein inhibitor, can selectively induce the death of senescent cells by activating the mitochondrial apoptosis pathway, and is one of the most widely studied senolytic drugs. However, ABT-263 has poor water solubility, a short half-life in vivo, and lacks the ability to actively target senescent cells. Furthermore, existing senolytic strategies only focus on the elimination of senescent cells themselves, without considering the simultaneous elimination of upstream pathogenic factors leading to senescence (such as senescent bacteria), making it difficult to fundamentally block the continuous production of senescent cells.

[0005] Biomimetic cell membrane-coated nanotechnology offers a novel approach to addressing the problem of insufficient targeting. By coating the surface of a nanocarrier with the source cell membrane, recognition molecules on the membrane surface can be transferred to the nanomedicine, endowing it with natural affinity for homologous cells and the ability to evade immune responses. However, there are currently no reports on constructing biomimetic nanocarriers using senescent tumor cell membranes for homologous targeted elimination of senescent tumor cells.

[0006] Therefore, developing a multifunctional nanomedicine delivery system that can simultaneously target senescent tumor cells, selectively eliminate senescent cells, kill senescent bacteria, and enhance chemotherapy sensitization is of great scientific significance and potential application value. Summary of the Invention

[0007] Technical problems to be solved

[0008] This invention aims to provide a biomimetic hybrid membrane liposome nanoformulation, its preparation method, and its applications, in order to at least partially solve the following problems:

[0009] (1) Senescent tumor cells are less sensitive to chemotherapy and are difficult to eliminate effectively;

[0010] (2) Tumor-associated bacterial factors may continuously induce / maintain aging and drug resistance phenotypes;

[0011] (3) Senolytic drugs and antibacterial drugs have defects such as uneven distribution, off-target effects and insufficient synergy when administered in free form;

[0012] (4) Existing cell membrane biomimetic systems have insufficient ability to enrich senescent tumor cells or lack a functional closed loop of "anti-senescence + antibacterial".

[0013] Technical solution

[0014] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0015] (1) This invention provides a biomimetic hybrid liposome nanomedicine, wherein the nanomedicine comprises a biomimetic hybrid vesicle structure formed by fusing liposomes co-loaded with a Bcl-2 / Bcl-xL inhibitor and an antibiotic with the membrane of senescent tumor cells; wherein the Bcl-2 / Bcl-xL inhibitor is distributed in the lipid bilayer, and the antibiotic is encapsulated in the aqueous core of the liposome. The Bcl-2 / Bcl-xL inhibitor selected is ABT-263 (Navitoclax) and the fluoroquinolone antibiotic ciprofloxacin.

[0016] The lipid composition of the liposomes includes phospholipids and cholesterol. The phospholipids are selected from one or more of egg yolk lecithin, soybean lecithin, dipalmitoylphosphatidylcholine, and distearate phosphatidylcholine; the mass ratio of phospholipid (EPC) to cholesterol (Chol) is 10:3. EPC:CIP (w:w) = 8:1, EPC:ABT-263 = 15:1; mouse colon cancer cells CT26 were used as a model. The obtained nanomedicine particles have a diameter around 180 nm. Furthermore, the encapsulation efficiency of ABT-263 is >90%, and the encapsulation efficiency of CIP is >75%.

[0017] (2) A method for preparing biomimetic hybrid liposome nanomedicines, comprising the following steps:

[0018] (a) Preparation of co-loaded liposomes: Phospholipids, cholesterol and Bcl-2 / Bcl-xL inhibitors were dissolved in an organic solvent, and the organic solvent was removed by vacuum drying to form a lipid film. The lipid film was hydrated with an aqueous solution containing antibiotics to obtain a crude liposome dispersion. After ultrasonic treatment, the dispersion was extruded through a polycarbonate membrane and then the free drug was removed by ultrafiltration and centrifugation to obtain co-loaded liposomes.

[0019] (b) Extraction of senescent tumor cell membranes: Tumor cells were induced to age by chemotherapy drugs, collected, lysed in hypotonic atmosphere, homogenized in an ice bath, and separated by differential centrifugation to obtain cell membrane components.

[0020] (c) Preparation of biomimetic hybrid liposomes: The liposomes obtained in step (a) are mixed with the cell membranes obtained in step (b) in a certain proportion, and after ultrasonic treatment, they are repeatedly extruded through a liposome extruder to obtain biomimetic hybrid liposome nanomedicines.

[0021] (3) Used to clear senescent tumor cells and inhibit tumor-associated bacteria, thereby enhancing the anti-tumor effect of chemotherapy drugs; the chemotherapy drugs preferably include 5-FU.

[0022] Beneficial effects:

[0023] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0024] (1) By fusing the membranes of senescent tumor cells with liposome membranes to form a hybrid membrane, the nano-preparation can enhance the uptake of homologous senescent tumor cells, increase the local effective concentration, and reduce the non-specific uptake tendency in normal cells.

[0025] (2) Co-load senolytic drugs and antibacterial drugs on the same nanoplatform to achieve simultaneous intervention of clearing senescent tumor cells and inhibiting bacteria;

[0026] (3) The resulting formulation has a small particle size, good dispersibility, and certain storage stability under low temperature conditions;

[0027] (4) When used in combination with chemotherapy drugs, it can further reduce the vitality of senescent tumor cells and has the potential to enhance the sensitivity of chemotherapy. Attached Figure Description

[0028] Figure 1 Image showing the results of SA-β-Gal staining in DOX-induced senescent cells;

[0029] Figure 2 Figure: Results of DOX-induced EdU incorporation experiment in senescent cells;

[0030] Figure 3 : Particle size and PDI distribution of nano-formulations;

[0031] Figure 4 TEM image of nano-formulation (vesicle bilayer structure);

[0032] Figure 5 : FRET verification of membrane fusion results;

[0033] Figure 6 SDS-PAGE membrane protein banding;

[0034] Figure 7 Comparison of fluorescence uptake quantification in different cell lines;

[0035] Figure 8 : Graph showing the results of the antibacterial experiment;

[0036] Figure 9 Cell viability results of combining nanomedicine with 5-FU. Detailed Implementation

[0037] The present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the experimental materials, reagents and conditions used in the embodiments can be obtained or implemented by those skilled in the art using conventional technical means.

[0038] Example 1: Obtaining Senescent Tumor Cells

[0039] CT26 tumor cells were cultured to an appropriate density and then treated with a medium containing doxorubicin (DOX) to induce cell senescence. The preferred conditions were: continuous treatment with 1 μM DOX for 5 days. The senescence phenotype was confirmed by SA-β-Gal staining and EdU incorporation assays.

[0040] Example 2: Extraction of senescent tumor cell membrane (SCCM)

[0041] Senescent cells obtained in Example 1 were collected, washed with PBS, and centrifuged to collect the cell pellet. Lysis buffer containing the protease inhibitor PMSF (final concentration 1 mM) was added, and the cells were lysed in an ice bath for about 15 min. The lysis buffer was then placed in a pre-chilled glass homogenizer for homogenization and disruption, about 40 times. About 5 μL of sample was taken for observation of cell disruption under a microscope. After the cells were basically disrupted, the centrifugation step was performed.

[0042] The homogenate was subjected to differential centrifugation: first, the cell nuclei and unbroken cells were removed by low-speed centrifugation (e.g., 700 g, 4 ℃, 10 min), and the supernatant was collected; then, the membrane component precipitate was collected by high-speed centrifugation (e.g., 14000 g, 4 ℃, 30 min), and the precipitate was retained to obtain senescent tumor cell membrane component (SCCM), which was stored at -80 ℃ for later use.

[0043] Example 3: Preparation of ABT-263 / CIP·HCl co-loaded liposomes (thin-film hydration method)

[0044] Weigh out egg yolk lecithin (EPC) and cholesterol (Chol) at a ratio of EPC:Chol = 10:3, with the total EPC amount controlled at 20 mg. Add 3 mL of dichloromethane (DCM) and dissolve thoroughly. Then add ABT-263 to make the ABT-263:EPC ratio 1:15 (w / w). Mix well and transfer to a vial. Place the mixture under vacuum drying conditions (e.g., 35 °C) to evaporate the solvent and form a uniform lipid film. Continue drying to remove residual solvent.

[0045] Ciprofloxacin (CIP) was dissolved in deionized water to prepare an aqueous phase, with a fixed hydration volume of 3 mL, resulting in a CIP:EPC ratio of 1:8 (w / w). This aqueous phase was then added to the lipid film and hydrated at 37 °C and 100 rpm for 60 min to form a crude liposome dispersion.

[0046] The dispersion was sonicated in an ice bath to reduce the particle size (e.g., 25% power, 1 s sonication / 3 s interval, 5 min total). It was then extruded sequentially through polycarbonate membranes with 400 nm and 200 nm pore sizes, with the 400 nm membrane being extruded 15 times and the 200 nm membrane being extruded 15 times, to obtain ABT-263 / CIP co-loaded liposomes (AC@lip) with a relatively uniform particle size distribution.

[0047] Example 4: Removal of free drug (one ultrafiltration cycle)

[0048] The obtained liposome solution was added to an ultrafiltration tube with a molecular weight cutoff of 100 kDa, centrifuged at 2000 g and 4 °C for 10 min, and the filtrate was discarded. The liposome solution retained in the ultrafiltration tube was collected for subsequent membrane fusion and characterization.

[0049] Example 5: Preparation of biomimetic hybrid membrane liposome nanoformulations (membrane fusion)

[0050] The resulting co-loaded liposomes were mixed with the SCCM obtained in Example 2 at a mass ratio of 1:1 (w / w), and the mixture was subjected to probe ultrasound under ice bath conditions to promote membrane fusion. The ultrasound parameters were: power 25%, work / pause mode of 3 s on, 7 s pause, and a total working time of 5 min. Subsequently, the mixture was further extruded using a liposome extruder to form 400 nm and 200 nm carbonate membranes, respectively, to obtain a biomimetic hybrid membrane liposome nanoformulation (e.g., AC@HM(lip-SCCM)).

[0051] Example 6: Physicochemical Characterization and Stability

[0052] The obtained hybrid membrane liposome nanoformulation was characterized. The results showed an average particle size of approximately 157.6 nm, a PDI < 0.3, and a Zeta potential of approximately -0.5 mV. Transmission electron microscopy revealed a distinct vesicular bilayer structure. After storage at 4 °C for 7 days, the particle size fluctuated by approximately 20 nm, indicating good storage stability.

[0053] Example 7: HPLC determination method

[0054] ABT-263 and CIP·HCl were quantitatively detected by HPLC. The chromatographic conditions were as follows:

[0055] Chromatographic column: C18 reversed-phase column (e.g., Waters, 150 mm × 4.6 mm, 5 μm);

[0056] Mobile phase: methanol-0.2% phosphoric acid aqueous solution (pH=3.0), gradient elution;

[0057] Gradient procedure:

[0058] Time (min) Methanol (%) 0.2% phosphoric acid solution (%) 0 30 70 3 40 60 6 60 40 11 70 30 15 100 0 16 100 0

[0059] Flow rate: 1.0 mL / min; column temperature: 30 ℃; injection volume: 10 μL; detection wavelength: 278 nm; run time: 16 min.

[0060] Under the above conditions, the CIP retention time is approximately 6.2 min, and the ABT-263 retention time is approximately 14.4 min.

[0061] Example 8: Standard Curve

[0062] The standard curve is established using the external standard method, where:

[0063] ABT-263 standard curve: Y = 10980X − 19034, R² = 0.9942; linear range: X = 5 ~ 160 μg / mL.

[0064] CIP standard curve: Y = 82675X + 17083, R² = 0.9998; linear range: X = 5~160 μg / mL.

[0065] X represents μg / mL, and Y represents the peak area.

[0066] Example 9: Determination of encapsulation efficiency (EE%) and drug loading (LE%) (based on liposome stage testing)

[0067]

[0068]

[0069] In a preferred embodiment of the present invention, the drug loading and encapsulation efficiency are mainly characterized by the liposome stage detection results; drug leakage is small during the membrane fusion process, so the change in drug loading level after hybridization is not significant.

[0070] Example 10: Optimization of ABT-263 Feed Ratio

[0071] The effect of the EPC to ABT-263 mass ratio on encapsulation efficiency and drug loading was investigated. The results are as follows:

[0072] EPC: ABT-263 (w: w) EE% LE% 15:1 91.06 4.46 20:1 94.55 4.62 25:1 76.90 3.79 30:1 67.75 3.36

[0073] Example 11: Optimization of CIP Feed Ratio

[0074] The effect of the EPC to CIP mass ratio on encapsulation efficiency and drug loading was investigated. The results are as follows:

[0075] EPC: CIP (w: w) EE% LE% 6:1 66.4 7.84 8:1 77.26 6.91 10:1 85.78 6.19 12:1 75.02 4.61

[0076] Example 12: Cross-combination experiment

[0077] Based on the above screening results, cross-combination experiments were conducted using EPC:CIP·HCl ratios of 8:1 and 10:1, and EPC:ABT-263 ratios of 15:1 and 20:1, with groups 1, 2, 3, and 4 set up.

[0078] Group EPC: CIP (w: w) EPC: ABT-26 (w: w) ABT-263-EE% ABT-263-LE% CIP-EE% CIP-LE% 1 10:1 15:1 90.58 4.17 84.88 5.84 2 10:1 20:1 96.40 3.35 87.81 6.12 3 8:1 15:1 96.48 4.35 81.37 6.94 4 8:1 20:1 91.46 3.17 84.46 7.27

[0079] Ultimately, group A was selected as the optimal feed ratio: EPC:CIP·HCl = 8:1 (w / w) and EPC:ABT-263 = 15:1 (w / w). Under these conditions, the drug loading of the resulting liposomes was CIP-LE% = 6.94% and ABT-263-LE% = 4.35%, respectively, and the encapsulation efficiencies were CIP-EE% = 81.37% and ABT-263-EE% = 96.48%, respectively.

[0080] Example 13: Comparison of fluorescence quantification of uptake by different cell lines

[0081] Fluorescently labeled hybrid membrane nanoparticles were co-incubated with different cell lines for 4 hours before quantitative fluorescence analysis was performed.

[0082] The results showed that, compared with the control formulation, senescent CT26 cells (SCT26) exhibited approximately 8.7-fold increased uptake of the nano-formula, while normal CT26 cells showed approximately 4.6-fold increased uptake; non-homologous tumor cells B16 and 4T1 showed approximately 3.4-fold and 3.7-fold increased uptake, respectively; and normal fibroblast L929 showed approximately 1.9-fold increased uptake. These results indicate that a hybrid membrane formed using senescent tumor cell membranes can significantly enhance uptake of homologous senescent tumor cells and reduce the tendency for non-specific uptake in normal cells.

[0083] Example 14: In vitro antibacterial experiment

[0084] Take Escherichia coli and add gradient concentrations of ABT-263 solution, ciprofloxacin solution, AC@lip and AC@HM (lip-SCCM). After incubation at 37°C for an appropriate time, evaluate the antibacterial effect by OD600 measurement and / or plate count.

[0085] Results: ABT-263 itself had no antibacterial activity; ciprofloxacin, AC@lip and AC@HM (lip-SCCM) all showed concentration-dependent antibacterial effects, and the hybrid membrane coating did not weaken their antibacterial efficacy.

[0086] Example 15: Synergistic Effect Experiment with 5-FU

[0087] SCT26 cells were seeded in 96-well plates, and the following experimental groups were set up:

[0088] 5-FU monotherapy group: 5-FU concentrations of 0, 10, 20, 30, 40, 50, and 60 µM;

[0089] Combination therapy group: Based on the above 5-FU concentrations, AC@HM (lip-SCCM) (50 nM or 100 nM based on ABT-263 concentration) was added respectively.

[0090] Cell viability was detected by CCK-8 assay after 48 h of culture.

[0091] Results: Cell viability decreased continuously with increasing 5-FU and nanomedicine concentrations. The cell-killing effect of the combined treatment group was significantly enhanced compared with both the 5-FU monotherapy group and the nanomedicine monotherapy group, showing a synergistic effect.

Claims

1. A biomimetic hybrid membrane liposome nanoformulation, characterized in that, include: The liposome core, a first active ingredient and a second active ingredient loaded in the liposome core, and a hybrid membrane covering the surface of the liposome core; the hybrid membrane is formed by the fusion of senescent tumor cell membrane and liposome membrane; the first active ingredient is a drug for clearing senescent cells, and the second active ingredient is an antibacterial drug.

2. A biomimetic hybrid liposome nanomedicine, characterized in that, The senescent tumor cell membrane is derived from senescent tumor cells induced by chemotherapy drugs; the chemotherapy drugs are selected from one of doxorubicin, cisplatin, and etoposide, with an induction concentration of 1 μM and an induction time of 5 days.

3. A method for developing biomimetic hybrid membrane liposome nanoformulations, characterized in that: Senescent tumor cell membranes were extracted; liposomes co-loaded with a first active ingredient and a second active ingredient were prepared using a thin-film hydration method. Specifically, phospholipids, cholesterol, and the first active ingredient were dissolved in dichloromethane to form a thin film, followed by hydration with an aqueous phase containing the second active ingredient to obtain liposomes. The liposomes obtained from the senescent tumor cell membranes were then fused through ultrasound and extrusion to form a hybrid membrane, resulting in a biomimetic hybrid membrane liposome nanoparticle formulation. The extrusion pore sizes included 400 nm and 200 nm, with the 400 nm membrane extruded 10–20 times and the 200 nm membrane extruded 10–20 times, preferably 15 times for each.

4. The use of the nano-formulation in the preparation of a drug, wherein the drug is used to eliminate senescent tumor cells and inhibit tumor-associated bacteria, thereby enhancing the antitumor effect of chemotherapy drugs.

Images