Ascorbyl palmitate nanoliposome capable of inducing tumor cell oncosis, and preparation method and application thereof

Abstract

The application relates to the field of biological medicine, and discloses ascorbyl palmitate nanoliposomes capable of inducing tumor cell oncosis, a preparation method and application thereof. The nanoliposomes are constructed by inserting ascorbyl palmitate as a lipid component into a phospholipid bilayer, and are prepared by a film dispersion method from lecithin, cholesterol, pegylated phospholipid and ascorbyl palmitate. The average particle size of the nanoliposomes is 80-150 nm, the encapsulation rate is high, and the stability is good. The nanoliposomes can be administered through a tail vein, utilize the specific hydrolysis of matrix metalloproteinase MMP-2 which is highly expressed by tumor cells to hydrolyze ascorbyl palmitate, release palmitic acid and ascorbic acid, thereby inducing Caspase-independent oncosis of tumor cells, effectively overcoming apoptosis-related drug resistance, and reversing a tumor immunosuppressive microenvironment. The nanoliposomes avoid the cumulative toxicity of metal-type inducers, and provide a new safe and effective option for reversing chemotherapy resistance.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an ascorbate palmitate nanoliposome that can induce tumor cell apoptosis, its preparation method, and its application. Background Technology

[0002] Studies have shown that the main mechanism by which chemotherapy kills tumor cells is the induction of apoptosis. However, tumor cells can develop chemotherapy resistance through various mechanisms, such as blocking apoptosis pathways, activating protective autophagy, and increasing drug efflux, which is one of the most important clinical obstacles in current cancer treatment. Furthermore, apoptotic cells can be promptly eliminated through the cytotoxic effects of macrophages, a process accompanied by the release of large amounts of immunosuppressive factors, which worsens the immunosuppressive nature of the tumor microenvironment, creates a "sanctuary" conducive to tumor recurrence and metastasis, and severely weakens the efficacy of chemotherapy. Non-apoptotic programmed cell death differs from apoptosis in terms of cell ultrastructure, signaling pathway regulatory proteins, and key proteins that execute cell death, providing a theoretical basis for circumventing apoptosis-induced drug resistance.

[0003] Oncosis is a caspase-independent, pormin-mediated, programmed cell death characterized by increased cell volume, bubbling of the cell membrane, swelling of organelles, cell membrane rupture, and subsequent leakage of cell contents, releasing damage-associated molecules such as heat shock 70kDa protein, calreticulin, and high-mobility group box 1 (HMP-1). More importantly, rapid mitochondrial dysfunction during oncosis leads to ATP depletion, thereby inhibiting ABC transporter activity and suppressing drug efflux. The caspase-independent nature of oncosis bypasses apoptosis-related resistance, significantly overcoming multidrug resistance and activating innate and adaptive immunity by promoting dendritic cell maturation, awakening adaptive cytotoxic T lymphocytes, and inducing macrophage polarization towards the M1 phenotype. Therefore, inducing oncosis to bypass apoptosis pathway blockade and restart tumor cell killing is of great significance for reversing chemotherapy resistance.

[0004] In preclinical studies, metal complexes (such as platinum, ruthenium, and copper complexes) have been widely explored due to their effectiveness in inducing tumor cell apoptosis; however, their cumulative toxicity has become a key bottleneck limiting clinical translation. L-ascorbic acid fatty acid esters are widely used in the oil, oily food, cosmetic, and pharmaceutical industries as antioxidants and surfactants. Among them, ascorbate palmitate (PA) is a lipid-soluble derivative formed by the ester bond between ascorbic acid (Asc) and palmitic acid. Subsequent studies have found that low doses of PA can induce apoptosis in various tumor cell types. However, if apoptosis is induced indiscriminately in both normal and tumor cells, it may cause serious side effects. Therefore, exploring novel agents that can induce cell apoptosis is of great significance for reversing chemotherapy resistance. Summary of the Invention

[0005] To obtain a novel formulation that can induce cell apoptosis, this invention provides an ascorbate palmitate nanoliposome that can induce tumor cell apoptosis, its preparation method, and its application. The liposomes prepared by this method avoid the limitations of clinical translation caused by metal accumulation toxicity and can be used to reverse chemotherapy resistance, providing a new option for tumor treatment.

[0006] The solution of the present invention is: An ascorbate palmitate nanoliposome that can induce tumor cell apoptosis, the nanoliposome comprising phospholipids, cholesterol, polyethylene glycol-modified phospholipids and ascorbate palmitate, wherein the ascorbate palmitate is inserted as a lipid component into the phospholipid bilayer; the average hydrated particle size of the nanoliposome is 80 nm to 150 nm, the polydispersity index is less than 0.3, and the encapsulation efficiency of the ascorbate palmitate is not less than 80%.

[0007] As a preferred technical solution, the mass ratio of phospholipids, cholesterol and polyethylene glycol-modified phospholipids is 70-225:17.5-56.5:5-10.

[0008] As a preferred technical solution, the mass ratio of ascorbate palmitate to phospholipid is 5-75:70-225.

[0009] As a preferred technical solution, the polyethylene glycol-modified phospholipid is DSPE-PEG2000.

[0010] As a preferred technical solution, the average hydrated particle size of the obtained nanoliposomes is 100 nm to 120 nm; the polydispersity index is less than 0.2; and the encapsulation efficiency of the ascorbate palmitate is not less than 90%.

[0011] This invention also discloses a method for preparing nanoliposomes that can induce tumor cell apoptosis, comprising the following steps: S1. Dissolve phospholipids, cholesterol, and polyethylene glycol-modified phospholipids in chloroform and sonicate to ensure complete dissolution; S2. Add ascorbate palmitate to the solution obtained in step S1 and sonicate to dissolve it completely. S3. The solution obtained in step S2 is rotary evaporated in a water bath at 45 ℃~48 ℃ for 40~60 min to remove the organic solvent and form a lipid film. S4. Add hydration medium to the lipid film obtained in step S3, incubate at 45 °C and 150–200 rpm for 1 h, and then sonicate under ice bath conditions to obtain an emulsion. S5. Dialyze the emulsion obtained in step S4 to remove free ascorbate palmitate and raw materials, thereby obtaining the nanoliposomes.

[0012] As a preferred technical solution, the amount of chloroform used is such that the concentration of phospholipid in chloroform is in the range of 5 mg / mL to 20 mg / mL.

[0013] As a preferred technical solution, the ultrasound time in step S1 is 15 to 30 minutes.

[0014] As a preferred technical solution, the amount of ascorbate palmitate added in step S2 is 5-75 mg; the ultrasonic time is 15-30 min.

[0015] As a preferred technical solution, the volume of the hydration medium in step S4 is 5 to 6 mL.

[0016] As a preferred technical solution, the ultrasonic treatment in step S4 has a power of 200 W and adopts a cycle of 3 seconds of ultrasound followed by 5 seconds of intermittent ultrasound, for a total of 20 cycles.

[0017] The present invention also discloses the application of a nanoliposome that can induce tumor cell apoptosis in the preparation of a drug for inducing tumor cell apoptosis.

[0018] As a preferred technical solution, the tumor cells include, but are not limited to, colorectal cancer cells, melanoma cells, breast cancer cells, or cervical cancer cells.

[0019] This invention also discloses the application of a nanoliposome that can induce tumor cell apoptosis in the preparation of a tumor therapeutic drug for reversing chemotherapy resistance.

[0020] Compared with the prior art, the advantages of the present invention are: (1) The present invention provides a method for preparing nanoliposomes that can induce tumor cell apoptosis. The method is simple, safe and convenient. The nanoliposomes that can induce cell apoptosis have good stability and uniform particle size.

[0021] (2) The nanoliposomes of the present invention that can induce cell apoptosis can respond to the disintegration of matrix metalloproteinases highly expressed in tumor cells, thereby achieving specific activation of the tumor microenvironment.

[0022] (3) The nanoliposomes provided by the present invention can effectively induce tumor cell apoptosis and overcome chemotherapy resistance related to apoptosis pathway; the tumor cells include, but are not limited to, colorectal cancer cell line MC38, melanoma cell line B16-F10, breast cancer cell line 4T1, and cervical cancer cell line HeLa.

[0023] (4) In the experiment of this invention, after the liposome enters the body through the tail vein, PA, as a lipid-soluble molecule, can preferentially enter tumor cells with active lipid metabolism through passive diffusion. After entering the cell, PA is decomposed into palmitic acid and ascorbic acid under the action of the matrix metalloproteinase MMP-2 highly expressed in tumor cells, inducing tumor cell apoptosis. It can be used to treat a variety of tumors; and it can produce a synergistic effect with chemotherapy drugs, improve the anti-tumor effect, and show good biocompatibility. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0025] Figure 1 The images show transmission electron microscopy (TEM) images and particle size changes during a 7-day stability study of the PA liposomes in Example 1 of this invention; where A is the TEM image and B is the particle size change diagram. Figure 2 These are characterization diagrams of cell morphology and cell membrane changes induced by PA in various cell apoptosis in Example 2 of the present invention; wherein, A is a cell morphology diagram and B is a characterization diagram of the changes; Figure 3 This is an H&E staining image of a mouse colon tumor section treated with PA liposomes in vivo during Example 3 of the present invention. Figure 4 This is a graph showing the change in tumor volume in mice during in vivo antitumor treatment with PA liposomes in Example 3 of the present invention. Figure 5 This is a graph showing the change in mouse body weight during in vivo antitumor treatment with PA liposomes in Example 3 of the present invention. Detailed Implementation

[0026] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0027] Example 1: Preparation of PA liposomes that can induce tumor cell apoptosis This embodiment provides a method for preparing PA liposomes (Lipo / PA).

[0028] (1) Weigh 225 mg of lecithin, 75 mg of cholesterol and 10 mg of DSPE-PEG-2000 and add them to a 100 mL round bottle. Then add 15 mL of chloroform and sonicate for 30 min to dissolve them completely. (2) Weigh 50 mg PA and add it to the above-mentioned eggplant-shaped flask, and sonicate for 30 min to dissolve it completely; (3) The solution obtained in (2) was rotary evaporated at 45 °C for 50 min to completely remove the organic solvent chloroform. A uniform light yellow film could be observed at the bottom of the flask. (4) Add 5 mL of hydration medium to the (3) eggplant-shaped flask, incubate at 180 rpm and 45 ℃ for 1 h, and then perform ice bath sonication treatment using an ultrasonic cell disruptor at 200 W power with a 3 s sonication interval, 5 s interval, and 20 cycles to obtain a homogeneous emulsion. (5) Transfer the emulsion obtained in (4) into a dialysis bag and dialyze for 24 h to remove free ascorbate palmitate and raw materials, thereby obtaining the nanoliposome Lipo / PA, which is stored at 4 °C for later use.

[0029] The PA liposomes prepared by the above method were characterized by transmission electron microscopy. The liposomes were then incubated at room temperature for 7 days, and their particle size and dispersion index (PDI) were measured daily. The results are as follows: Figure 1 As shown, the prepared liposomes are regular spherical with a uniform particle size distribution. Based on electron microscopy scale bars, their main distribution is estimated to be around 100 nm. Stability testing results showed that neither the particle size nor the PDI changed significantly within 7 days, indicating that Lipo / PA has good storage stability. Its average particle size is approximately 100-120 nm, PDI is less than 0.2, and encapsulation efficiency is greater than 90%.

[0030] Example 2: Evaluation of PA liposome-induced apoptosis in various tumor cells In this embodiment, PA liposomes were applied to various tumor cell lines (MC38, 4T1, B16F10, HeLa) to evaluate their ability to induce cell apoptosis.

[0031] The operational evaluation steps are as follows: Cells in the logarithmic growth phase are divided into groups of 2 × 10⁻⁶. 5The cells were seeded into six-well plates and cultured in an incubator for 24 hours. The old culture medium was then discarded, and blank culture medium and culture medium containing PA liposomes were added respectively. After incubation for a certain period of time, the cell morphology was observed under a microscope to check for cell bloating and apoptosis, and the results were recorded by taking pictures.

[0032] At the same time, cells in the logarithmic growth phase were divided into groups of 1.5 × 10⁻⁶. 5 The cells were seeded into confocal microscopy dishes and cultured in an incubator for 24 h. The old culture medium was discarded, and blank culture medium and drug-containing culture medium were added. The cells were incubated in an incubator for 4 h, then the culture medium was discarded. The cells were washed twice with PBS solution, and 1 mL of cell membrane staining working solution (prepared with culture medium to a final concentration of 10 μM) was added. The cells were gently shaken to ensure even staining of all cells. The cells were incubated at 37 ℃ in the dark for 15 min. The cell membrane staining working solution was removed, and 0.5 mL of pre-chilled 75% ethanol was added to each well to fix the cells for 10 min. The ethanol was then removed, and the cells were washed three times with PBS solution. 1 mL of 1 μg / mL DPAI working solution was added to each well, and the cells were incubated for 10 min. The working solution was then removed, and the cells were washed three times with PBS solution. The cells were observed and photographed using a confocal microscope.

[0033] like Figure 2 As shown, various tumor cell types (MC38, 4T1, B16F10, and HeLa) exhibited significant morphological changes characteristic of apoptosis, such as cell swelling and cell membrane bulging and bubbling, after treatment with PA liposomes. This demonstrates that the PA liposomes prepared in this invention can effectively induce apoptosis in various tumor cell types.

[0034] Example 3: Application of PA liposomes in the treatment of colorectal cancer This embodiment evaluates the efficacy of PA liposomes in the in vivo treatment of colorectal cancer and their synergistic effect with the chemotherapy drug oxaliplatin (OXP).

[0035] Evaluation site: A subcutaneous MC38 tumor model was constructed using 6-8 week old C57BL / 6 female mice. First, the axillary hair of the mice was shaved. Then, MC38 cells in the logarithmic growth phase and in good growth condition were injected at a rate of 2 × 10⁻⁶. 6 Cells were inoculated subcutaneously into the right axilla of mice at a dose of 100 cells / mouse. Tumor growth was observed daily, and the length (a) and width (b) of the tumor were measured with calipers. The tumor volume (V) was calculated: V (mm²) 3 ) = (a × b 2 ) / 2, when the tumor volume reaches 100 mm 3 When the mouse model was successfully established (V=80 mm), it was considered to have been successfully modeled. 3 ~120 mm 3Mice were randomly divided into five groups: ① Control (saline), ② Blank Lipo (blank liposomes), ③ Lipo / PA, ④ Lipo / OXP (oxaliplatin liposomes), and ⑤ Lipo / PA / OXP (co-loaded liposomes). Six mice were in each group. Administered via tail vein every other day for two weeks, monitoring tumor volume and mouse weight throughout the treatment period. Initial weight and tumor volume were measured before administration. Following each administration, mice were weighed and their tumor volume was measured to record changes in tumor growth. Tumor tissue was collected after treatment and subjected to H&E staining for pathological analysis.

[0036] The results are as follows Figure 3 , 4 As shown in Figure 5: H&E staining results (e.g.) Figure 3 As shown in the figure, the tumor tissues of the blank control group and the Blank Lipo group showed obvious typical characteristics of malignant tumors: tightly packed cells, clear cell nuclei, and vigorous growth; the tumor tissues of the Lipo / OXP group and the Lipo / PA group showed necrotic areas and an increase in abnormally shaped cells; the Lipo / PA / OXP group showed changes in cell morphology, loose cell arrangement, and obvious large areas of necrotic tissue, indicating that PA can effectively enhance the anti-tumor effect of chemotherapy drugs.

[0037] Tumor volume changes (e.g.) Figure 4 As shown in the figure, the tumor volume of mice in the control group and Blank / Lipo group increased significantly over time, and the growth was rapid with no obvious inhibitory effect. The relative tumor volume of Lipo / OXP group and Lipo / PA group decreased. After Lipo / PA / OXP treatment, tumor growth was significantly inhibited due to the synergistic effect of PA and OXP, which improved the anti-tumor effect of OXP.

[0038] Weight change curve (e.g.) Figure 5 As shown in the figure, due to the toxic side effects of OXP, the body weight of mice in the Lipo / OXP group decreased during the administration period, while the body weight of mice in the other groups did not decrease significantly, indicating that Lipo / PA / OXP has good safety and biocompatibility.

[0039] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. An ascorbate palmitate nanoliposome that can induce tumor cell apoptosis, characterized in that: The nanoliposomes contain phospholipids, cholesterol, polyethylene glycol-modified phospholipids, and ascorbate palmitate, wherein the ascorbate palmitate is inserted into the phospholipid bilayer as a lipid component; the average hydrated particle size of the nanoliposomes is 80 nm to 150 nm, the polydispersity index is less than 0.3, and the encapsulation efficiency of the ascorbate palmitate is not less than 80%.

2. The ascorbate palmitate nanoliposome that can induce tumor cell apoptosis as described in claim 1, characterized in that: The mass ratio of the phospholipids, cholesterol and polyethylene glycol-modified phospholipids is 70–225:17.5–56.5:5–10.

3. The ascorbate palmitate nanoliposome that can induce tumor cell apoptosis as described in claim 1, characterized in that: The mass ratio of ascorbate palmitate to phospholipid is 5-75:70-225.

4. The ascorbate palmitate nanoliposome that can induce tumor cell apoptosis as described in claim 1, characterized in that: The PEGylated phospholipid is DSPE-PEG2000.

5. A method for preparing nanoliposomes capable of inducing tumor cell apoptosis as described in any one of claims 1 to 4, characterized in that, Includes the following steps: S1. Dissolve phospholipids, cholesterol, and polyethylene glycol-modified phospholipids in chloroform and sonicate to ensure complete dissolution; S2. Add ascorbate palmitate to the solution obtained in step S1 and sonicate to dissolve it completely. S3. The solution obtained in step S2 is rotary evaporated in a water bath at 45 ℃~48 ℃ for 40~60 min to remove the organic solvent and form a lipid film. S4. Add hydration medium to the lipid film obtained in step S3, incubate at 45 °C and 150–200 rpm for 1 h, and then sonicate under ice bath conditions to obtain an emulsion. S5. Dialyze the emulsion obtained in step S4 to remove free ascorbate palmitate and raw materials, thereby obtaining the nanoliposomes.

6. The method as described in claim 5, characterized in that: The amount of chloroform used is such that the concentration of phospholipids in chloroform is in the range of 5 mg / mL to 20 mg / mL.

7. The method as described in claim 5, characterized in that: The ultrasonic treatment in step S4 has a power of 200 W and uses a cycle of 3 seconds of ultrasound followed by 5 seconds of rest, for a total of 20 cycles.

8. The use of a nanoliposome capable of inducing tumor cell apoptosis as described in any one of claims 1 to 4 in the preparation of a medicament for inducing tumor cell apoptosis.

9. The application as described in claim 8, characterized in that, The tumor cells include, but are not limited to, colorectal cancer cells, melanoma cells, breast cancer cells, or cervical cancer cells.

10. The use of a nanoliposome capable of inducing tumor cell apoptosis as described in any one of claims 1 to 4 in the preparation of a tumor therapeutic agent for reversing chemotherapy resistance.

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