An ultrasonic response-based biomimetic epigenetic nanoregulator, a preparation method and application thereof

Abstract

The present application relates to a kind of based on ultrasonic response's biomimetic epigenetic nanoregulator and its preparation method and application, based on the biomimetic epigenetic nanoregulator of ultrasonic response includes: lipid nano core and hybrid biological membrane fused with it;The lipid nano core includes: liposome and load its lipid phase in acoustic sensitizer and load its water phase in DNA methyltransferase inhibitor;The hybrid biological membrane includes platelet membrane and with its membrane fusion thylakoid membrane.The biomimetic epigenetic nanoregulator is induced by ultrasonic irradiation immunogenic death of tumor cell, release a large number of damage associated molecular patterns, by regulating tumor cell epigenetic, up-regulate tumor cell surface class I histocompatibility complex (MHC-I) expression, increase MHC-I antigen peptide complex formation, can effectively improve the recognition of T cell to tumor cell, enhance anti-tumor immune response.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and relates to a biomimetic epigenetic nanoregulator based on ultrasound response, its preparation method and application, and particularly to a biomimetic epigenetic nanoregulator based on ultrasound response, its preparation method and its application in the preparation of antitumor drugs. Background Technology

[0002] Immunotherapy has become a powerful treatment for cancer; however, clinical trials have shown that only a small percentage of patients experience lasting benefit. This is primarily due to cancer's immune escape mechanisms. One reason is low levels of tumor antigens, resulting in insufficient ability of antigen-presenting cells (APCs) to present antigens to T cells. Additionally, in cancer types with low response and high resistance to anti-PD-1 / PD-L1 therapy (breast and colon cancer), downregulation of MHC-I expression prevents T cells from accurately recognizing tumor cells.

[0003] Recent reports indicate that epigenetic therapies such as DNA methyltransferase inhibitors (DNMTi) can not only upregulate the expression of tumor-associated antigens, but also enhance antigen presentation by inducing the enhancement of major histocompatibility complex (MHC) class I molecules on tumor cells through cancer testis antigen (CTA), induce dendritic cell (DC) maturation, thereby significantly affecting the tumor microenvironment (TME), suppressing immunosuppressive cells (MDSCs and Tregs), activating anti-tumor T effector cells, and ultimately improving the recognition and killing of tumor cells by T cells.

[0004] Epigenetic therapy can be further combined with other anticancer therapies (including immunotherapy, chemotherapy, radiotherapy, molecular targeted therapy, photothermal therapy, photodynamic therapy, photoacoustic imaging, etc.) to synergistically improve anti-tumor efficiency through different mechanisms. Sonodynamic therapy (SDT), due to its non-invasive and spatiotemporally controllable characteristics, has become the preferred adjuvant anti-tumor treatment. Combined therapy with SDT and epigenetic regulation can enhance the immunogenicity of cancer cells, strengthen the antigen-presenting ability of APCs, and reverse immunosuppressive tumor microenvironment (TME), thereby initiating a systemic anti-tumor immune response and effectively preventing tumor recurrence and metastasis by generating durable immune memory. Therefore, developing an effective strategy for treating tumors by combining sonodynamic therapy with epigenetic regulation is of great significance. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a biomimetic epigenetic nanoregulator based on ultrasound response, its preparation method, and its application, particularly relating to a biomimetic epigenetic nanoregulator based on ultrasound response, its preparation method, and its application in the preparation of antitumor drugs.

[0006] The biomimetic epigenetic nanomodulator induces immunogenic death of tumor cells through ultrasound irradiation, releasing a large number of damage-associated molecular patterns (DAMPs). By regulating the epigenetics of tumor cells, it upregulates the expression of class I histocompatibility complex (MHC-I) on the surface of tumor cells and increases the formation of MHC-I antigen peptide complexes, which can effectively improve the recognition of tumor cells by T cells and enhance the anti-tumor immune response.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a biomimetic epigenetic nanoregulator based on ultrasound response, wherein the biomimetic epigenetic nanoregulator based on ultrasound response comprises: a lipid nanocore and a hybrid biomembrane fused thereto;

[0009] The lipid nanocore comprises: a liposome and a sonosensitive agent loaded in its lipid phase and a DNA methyltransferase inhibitor loaded in its aqueous phase;

[0010] The hybrid biomembrane includes a platelet membrane and a thylakoid membrane fused to it.

[0011] This invention utilizes a phospholipid bilayer biomimetic nanoplatform and employs membrane fusion technology to construct a biomimetic epigenetic nanomodulator with a specific structure. The platelet membrane prevents the nanoplatform from being phagocytosed by macrophages, thus avoiding immune clearance and increasing blood circulation time. Simultaneously, it targets tumor sites through the action of p-selectin on platelets. The thylakoid membrane alleviates the hypoxia problem in the tumor immune microenvironment by catalyzing the generation of oxygen from hydrogen peroxide. Under ultrasound radiation, the sonosensitive agent converts oxygen into reactive oxygen species (ROS), which kill tumor cells. Simultaneously, under ultrasound radiation, the biomimetic epigenetic nanomodulator gradually decomposes and releases a DNA methyltransferase inhibitor, alleviating the immunosuppressive tumor microenvironment, promoting immunogenic tumor cell death, and enhancing the expression and presentation of the tumor surface antigen MHC-I. This recruits more cytotoxic T lymphocytes to infiltrate the tumor site, restoring the function of T cell-mediated immune responses. The synergistic effect of the sonosensitive agent and the DNA methyltransferase inhibitor can effectively inhibit the growth of tumors, including those resistant to anti-PD-1 drugs, induce long-term immune memory effects, and prevent tumor metastasis and recurrence. This provides a meaningful approach for the treatment of anti-PD-1 resistant tumors.

[0012] Preferably, the raw materials for preparing the liposomes include phospholipids, cholesterol, and DSPE-PEG.

[0013] Preferably, the phospholipid includes soybean lecithin.

[0014] Preferably, the number average molecular weight of PEG in the DSPE-PEG is 1000-4000, for example, 4000, 4000, 4000, 4000, 4000, 4000, 4000, etc.

[0015] Preferably, the sound-sensitizing agent includes hematoporphyrin monomethyl ether (which can be abbreviated as HMME in this invention).

[0016] Preferably, the DNA methyltransferase inhibitor includes zeblaline (which may be abbreviated as Zeb in this invention).

[0017] Preferably, the mass ratio of the liposome to the sonosensitive agent is (1-10):(0.1-1), wherein the specific values ​​in (1-10) can be selected as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., and the specific values ​​in (0.1-1) can be selected as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc.

[0018] Preferably, the lipid nanocore has a particle size of 100-130 nm, such as 105 nm, 110 nm, 120 nm, 125 nm, 128 nm, 130 nm, etc.

[0019] Preferably, the hybrid biofilm has a particle size of 130-150 nm, such as 130 nm, 135 nm, 140 nm, 142 nm, 145 nm, 150 nm, etc.

[0020] Preferably, the particle size of the biomimetic epigenetic nano-regulator is 170-200nm, such as 170nm, 175nm, 180nm, 185nm, 190nm, 200nm, etc.

[0021] Preferably, the mass ratio of the sound-sensitive agent to the DNA methyltransferase inhibitor is (1-10):(1.5-15), wherein the specific values ​​in (1-10) can be selected as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., and the specific values ​​in (1.5-15) can be selected as 1.5, 2, 4, 6, 8, 10, 12, 14, 15, etc.

[0022] Preferably, the protein concentration ratio of the platelet membrane to the thylakoid membrane is (1-5):(1-5), wherein the specific point values ​​in (1-5) can be selected as 1, 2, 3, 4, 5, etc.

[0023] Preferably, the mass ratio of the lipid nanocore to the hybrid biomembrane is (10-100):(1-10), wherein the specific values ​​in (1-10) can be selected as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., and the specific values ​​in (10-100) can be selected as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.

[0024] In a second aspect, the present invention provides a method for preparing a biomimetic epigenetic nanoregulator based on ultrasonic response according to the first aspect, the method comprising:

[0025] (1) A lipid film is obtained by rotary evaporation of an organic mixture containing liposome raw materials and a sound-sensitive agent;

[0026] (2) The DNA methyltransferase inhibitor solution was mixed with the lipid film and hydrated to obtain the lipid nanocore;

[0027] (3) The lipid nanocore was mixed with the hybrid biomembrane and co-incubated to obtain the ultrasound-responsive biomimetic epigenetic nanoregulator.

[0028] This invention utilizes a phospholipid bilayer biomimetic nanoplatform to construct a biomimetic epigenetic nanoregulator (hereinafter referred to as TPLHZ or TK / PLT@Lip-HMME / Zeb) via membrane fusion technology. First, a lipid nanocore (hereinafter referred to as Lip-HMME / Zeb) was synthesized as the nanoregulator using a thin-film dispersion method. Platelet membranes and thylakoid membranes were obtained separately by differential centrifugation. After co-incubation of the two membranes, a biomimetic hybrid membrane (hereinafter referred to as TK / PLT) was obtained. Co-incubation of the biomimetic hybrid membrane with the lipid nanocore formed a nanoscale drug delivery system, TPLHZ. This preparation method is applicable to industrial production, and the product obtained by this method exhibits excellent antitumor activity.

[0029] Preferably, the method for preparing the hybrid biomembrane includes: mixing and co-incubating platelet membrane and thylakoid membrane.

[0030] Preferably, the co-incubation is carried out at 30-45℃ (e.g., 30℃, 33℃, 35℃, 36℃, 37℃, 38℃, 40℃, 42℃, 43℃, 45℃, etc.) for 5-20 minutes (e.g., 5 minutes, 8 minutes, 10 minutes, 12 minutes, 15 minutes, 18 minutes, 20 minutes, etc.).

[0031] Preferably, ultrasonic-assisted treatment is also added during the co-incubation process.

[0032] Preferably, the platelet membrane is an autologous platelet membrane.

[0033] The platelet membrane and thylakoid membrane involved in this invention can be prepared using methods reported in the prior art or conventional techniques known in the art. For example, they can be prepared in the following manner:

[0034] Preferably, the platelet membrane is prepared by differential centrifugation, specifically including:

[0035] (S1) Centrifuge blood at 80-150g (e.g., 80g, 90g, 100g, 110g, 120g, 130g, 140g, 150g, etc.) for 10-40min (e.g., 15min, 20min, 30min, 35min, etc.).

[0036] (S2) Take the supernatant and centrifuge it at 80-150g (e.g., 80g, 90g, 100g, 110g, 120g, 130g, 140g, 150g, etc.) for 10-40min (e.g., 15min, 20min, 30min, 35min, etc.);

[0037] (S3) Take the supernatant and centrifuge it at 700-1000g (e.g., 700g, 7500g, 800g, 850g, 900g, etc.) for 10-40min (e.g., 15min, 20min, 30min, 35min, etc.) to collect platelets;

[0038] Preferably, the method for preparing the thylakoid membrane is differential centrifugation, specifically including:

[0039] (T1) After homogenizing the spinach, filter it and centrifuge the filtrate at 6000-8000 rpm (e.g., 6000 rpm, 6500 rpm, 7000 rpm, 7500 rpm, 8000 rpm, etc.) for 5-20 min (e.g., 5 min, 10 min, 15 min, 20 min, etc.).

[0040] (T2) Take the sediment and resuspend it, then centrifuge it at 10,000-14,000g (e.g., 10,000 rpm, 11,000 rpm, 12,000 rpm, 13,000 rpm, 14,000 rpm, etc.) for 20-40 min (e.g., 20 min, 25 min, 30 min, 35 min, 40 min, etc.).

[0041] (T3) Resuspend the sediment and centrifuge at 14,000-20,000 g (e.g., 14,000 rpm, 15,000 rpm, 16,000 rpm, 17,000 rpm, 18,000 rpm, 19,000 rpm, 20,000 rpm, etc.) for 10-25 min (e.g., 10 min, 15 min, 20 min, 25 min, etc.) to collect the thylakoid membrane.

[0042] Preferably, the rotary evaporation in step (1) is carried out at 45-60°C (e.g., 45°C, 50°C, 55°C, 60°C, etc.) for 30-180 min (e.g., 30 min, 50 min, 70 min, 80 min, 100 min, 120 min, 150 min, 180 min, etc.).

[0043] Preferably, the hydration in step (2) is carried out at 20-25°C (e.g., 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, etc.).

[0044] Preferably, the co-incubation in step (3) is carried out at 30-45℃ (e.g., 30℃, 32℃, 33℃, 35℃, 37℃, 40℃, 45℃, etc.) for 5-20 minutes (e.g., 5 minutes, 8 minutes, 12 minutes, 15 minutes, 16 minutes, 18 minutes, 20 minutes, etc.).

[0045] Preferably, ultrasonic-assisted treatment is also added during the co-incubation process.

[0046] In this invention, other specific point values ​​within all the numerical ranges mentioned above can be selected, but due to space limitations and for the sake of brevity, they will not be described in detail here.

[0047] Thirdly, the present invention provides the application of the ultrasound-responsive biomimetic epigenetic nanomodulators described in the first aspect in the preparation of antitumor drugs.

[0048] Preferably, the tumor includes anti-PD-1 resistant tumors.

[0049] The nanomodulators involved in this invention can effectively inhibit the growth of anti-PD-1 resistant tumors, induce long-term immune memory effects, and prevent tumor metastasis and recurrence.

[0050] Fourthly, the present invention provides the use of the ultrasound-responsive biomimetic epigenetic nanomodulator according to the first aspect in the preparation of formulations that upregulate the expression of MHC-I on the surface of tumor cells.

[0051] The biomimetic epigenetic nanoregulator involved in this invention can be made into a simple experimental formulation for exploring the physiological metabolic processes of tumor cells. The formulation claimed in this invention is not for eliminating the cause or lesion, but is an application in the preparation of a formulation that upregulates the expression of MHC-I on the surface of tumor cells for a non-therapeutic purpose.

[0052] Fifthly, the present invention provides a method for upregulating the expression of MHC-I on the surface of tumor cells, the method comprising: co-incubating tumor cells with an effective amount of the ultrasound-responsive biomimetic epigenetic nanoregulator described in the first aspect.

[0053] The method claimed in this invention does not directly target living human or animal bodies, but rather isolated tumor cells. It only protects the method of upregulating the expression of MHC-I on the surface of tumor cells (at the cellular level). Furthermore, this method is not a process of eliminating the cause or lesion, nor is it a treatment method for directly improving the health of human or animal bodies. Rather, it is intended for theoretical research on the physiological and metabolic behavior of tumor cells and for screening more drugs for treating tumors.

[0054] In a sixth aspect, the present invention provides an antitumor combination drug composition comprising the ultrasound-responsive biomimetic epigenetic nanomodulator of claim 1 and an antiPD-1 antibody.

[0055] Preferably, the combined pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0056] Preferably, the combined pharmaceutical composition of the present invention can be administered alone or in combination with excipients to form an appropriate dosage form for administration. The pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0057] Preferably, the combined pharmaceutical composition is a single compound preparation or a combination of two separate preparations.

[0058] Preferably, the combined pharmaceutical composition is a combination of two separate formulations, which are administered simultaneously or sequentially.

[0059] The combined drug composition can be a single compound preparation or a combination of two separate preparations; when it is a combination of two separate preparations, it can be administered simultaneously, alternately, or sequentially.

[0060] Preferably, the preparation is any pharmaceutically acceptable dosage form, such as tablets, powders, suspensions, granules, capsules, solutions, enemas, emulsions, etc.

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] This invention utilizes a phospholipid bilayer biomimetic nanoplatform and employs membrane fusion technology to construct a biomimetic epigenetic nanomodulator with a specific structure. The platelet membrane prevents the nanoplatform from being phagocytosed by macrophages, thus avoiding immune clearance and increasing blood circulation time. Simultaneously, it targets tumor sites through the action of p-selectin on platelets. The thylakoid membrane alleviates the hypoxia problem in the tumor immune microenvironment by catalyzing the generation of oxygen from hydrogen peroxide. Under ultrasound radiation, the sonosensitive agent converts oxygen into reactive oxygen species (ROS), which kill tumor cells. Simultaneously, under ultrasound radiation, the biomimetic epigenetic nanomodulator gradually decomposes and releases a DNA methyltransferase inhibitor, alleviating the immunosuppressive tumor microenvironment, promoting immunogenic tumor cell death, and enhancing the expression and presentation of the tumor surface antigen MHC-I. This recruits more cytotoxic T lymphocytes to infiltrate the tumor site, restoring the function of T cell-mediated immune responses. The synergistic effect of the sonosensitive agent and the DNA methyltransferase inhibitor can effectively inhibit the growth of tumors, including those resistant to anti-PD-1 drugs, induce long-term immune memory effects, and prevent tumor metastasis and recurrence. This provides a meaningful approach for the treatment of anti-PD-1 resistant tumors. Attached Figure Description

[0063] Figure 1 This is a schematic diagram of the preparation steps of the ultrasound-responsive epigenetic nanoregulator (TPLHZ) in Example 1;

[0064] Figure 2 These are particle size distribution diagrams for different nano-formulations;

[0065] Figure 3 These are Zeta potential diagrams for different nanoparticle formulations;

[0066] Figure 4 This is a graph showing the particle size stability results of the nano-regulator TPLHZ after incubation in PBS for 7 days.

[0067] Figure 5 This is a transmission electron microscope image of the nano-modifier TPLHZ;

[0068] Figure 6 This is a co-location diagram of the TK membrane and the PLT membrane;

[0069] Figure 7 It is a full-wavelength scan of the nano-regulator TPLHZ, the acoustic sensitizer HMME, the DNA methyltransferase inhibitor Zeb, and a physical mixture of HMME and Zeb;

[0070] Figure 8 This is a graph showing the blood compatibility test results of the nanocarrier TK / PLT@Lip and the nanomodifier TK / PLT@Lip-HMME / Zeb;

[0071] Figure 9The results of cell viability determination of 4T1 cells after 24 hours of treatment with different drugs using the MTT assay are shown in the figure (*P<0.05, **P<0.01, ***P<0.001, ****P<0.001, data are expressed as mean ± standard deviation, n=3).

[0072] Figure 10 The results of cell viability determination of CT26 cells after 24 hours of treatment with different drugs using the MTT assay are shown in the figure (*P<0.05, **P<0.01, ***P<0.001, ****P<0.001, data are expressed as mean ± standard deviation, n=3).

[0073] Figure 11 The graph shows the ROS levels of 4T1 cells (*P<0.05, **P<0.01, ***P<0.001, ****P<0.001, data are expressed as mean ± standard deviation, n=3);

[0074] Figure 12 This is a statistical graph showing the tumor volume results of each group of mice in test example 6;

[0075] Figure 13 This is a statistical graph showing the changes in body weight of mice in each group in test example 6;

[0076] Figure 14 This is a statistical graph showing the tumor volume results of each group of mice in test case 7. Detailed Implementation

[0077] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0078] Unless otherwise specified, all preparation steps below shall be carried out at room temperature (25°C).

[0079] Example 1

[0080] This embodiment provides a biomimetic epigenetic nanomodulator (abbreviated as TPLHZ or TK / PLT@Lip-HMME / Zeb), and the preparation method is illustrated in the schematic diagram below. Figure 1 As shown, the details are as follows:

[0081] (1) Preparation of biomimetic hybrid biomembrane (TK / PLT):

[0082] (1.1) Preparation of thylakoid membrane (TK):

[0083] (1.1.1) First, take 25g of fresh spinach leaves, wash them with distilled water, and store them in the dark at 4℃ for 12h.

[0084] (1.1.2) Subsequently, the spinach leaves were cut into small pieces and placed in 75 ml of HEPES buffer (10 mM, pH = 7.6) containing 5 mM MgCl2, 50 mM NaCl and 0.3 M sucrose. The mixture was then placed at 4 °C in the dark for 45 min.

[0085] (1.1.3) Next, the spinach homogenate was ground in an ice mortar. Then, the leaf fragments were filtered through 10 layers of cotton gauze, and the filtrate was centrifuged at 8000 rpm for 10 min to collect the intact chloroplasts.

[0086] (1.1.4) The sediment was resuspended in HEPES buffer (10 mM, pH = 8.0) and allowed to stand at 4 °C for 4 h. Then it was centrifuged at 12000 rpm for 30 min. The matrix extract was removed from the supernatant and the thylakoid-rich particles were collected.

[0087] (1.1.5) After washing twice with 10mM HEPES buffer, resuspend in distilled water and centrifuge at 16000rpm for 15min to obtain TK particles in dark green. Finally, TK was stored at -80℃.

[0088] (1.2) Preparation of platelet membrane (PLT):

[0089] (1.2.1) Fresh blood was collected from female BALB / c mice in EDTA anticoagulant tubes, centrifuged at 100×g at room temperature for 20 min, and platelet-rich plasma was separated and collected in the supernatant.

[0090] (1.2.2) Then, centrifuge at 100×g for 20 min to remove residual red blood cells. Add 1×PBS buffer (10mM, pH=7.4) to the supernatant, which contains 1mM EDTA and 2μM prostaglandin E1 (PGE1) to prevent platelet activation.

[0091] (1.2.3) Platelets were then collected by centrifugation at 800×g at room temperature for 20 min. The obtained platelet particles were resuspended in 1×PBS (0.01M, pH=7.4) containing 1mM EDTA and protease inhibitor and frozen at -80℃.

[0092] (1.3) Preparation of hybrid membranes:

[0093] After ultrasonically pulverizing TK and PLT for 5 min, the concentrations of TK and PLT membrane proteins were determined using a BCA protein kit. Then, they were mixed at a 1:1 weight ratio and incubated at 37°C for 10 min to obtain the final product.

[0094] (2) Preparation of nano-modifiers:

[0095] (2.1) Preparation of Lip-HMME / Zeb:

[0096] (2.1.1) Dissolve 1 mg of HMME in 2 mL of methanol. Add soybean lecithin, cholesterol, and DSPE-PEG. 2k The HMME was dissolved in dichloromethane solution at a mass ratio of 5:1:3 to form a 10 mg lipid solution. The HMME was then mixed with the lipid solution in a round-bottom flask and subjected to rotary evaporation. After the organic solvent evaporated in the rotary evaporator, a thin film (abbreviated as Lip-HMME) formed at the bottom of the flask.

[0097] (2.1.2) Then, 1 mL of PBS solution containing Zeb (1.5 mg / mL) was added to the lipid membrane and hydrated at room temperature to obtain the lipid nanocore Lip-HMME / Zeb.

[0098] (2.2) Preparation of TK / PLT@Lip-HMME / Zeb:

[0099] The lipid nanocore Lip-HMME / Zeb was added to the hybrid membrane solution prepared in (1.3) and incubated at 37°C for 10 min. The mixture was then ultrasonically broken to obtain a uniform nano-regulator TK / PLT@Lip-HMME / Zeb.

[0100] Test Example 1

[0101] The particle size and zeta potential of the prepared nano-modifier TK / PLT@Lip-HMME / Zeb were analyzed.

[0102] Small amounts of liposomes (blank liposomes without HMME and Zeb loading, prepared according to Example 1) (A), Lip-HMME (B), Lip-HMME / Zeb (C), and TK / PLT@Lip-HMME / Zeb (D) prepared in each step of Example 1 were taken and diluted with PBS. The particle size and polydispersity index (PDI) of the samples were determined using a particle size analyzer. The particle size was measured daily for 7 days over one week to assess the stability of the nanomaterials. The zeta potential was then measured using a potentiometer. The potentials of the liposomes, Lip-HMME, Lip-HMME / Zeb, and TK / PLT@Lip-HMME / Zeb were measured using the same method. The obtained data were processed and analyzed using GraphPad Prism 8.

[0103] The particle size analysis results for each sample group are as follows: Figure 2 As shown, this invention optimizes soybean phospholipids, cholesterol, and DSPE-PEG. 2kWith the addition of [amount], liposomes with a particle size of (125.9±2.9) nm were synthesized. With further modification, the hydrated particle size gradually increased, and the hydrated particle size of the nano-regulator TPLHZ was (185.4±10.4) nm.

[0104] The zeta potential detection results for each sample group are as follows: Figure 3 As shown, the potential of the liposomes was (-339.4±0.83) mV. With gradual loading of HMME, Zeb, and Lip-HMME / Zeb, the potential gradually increased to (-15.7±1.1) mV, with positive and negative values ​​representing the charge carried by the particles. The hybrid biomembrane is negatively charged; therefore, after co-incubation of the nanocore and the hybrid membrane, the potential of the nanocomposite decreased to (-22.9±1.4) mV, consistent with the expected results.

[0105] The particle size analysis results of the nano-regulator TPLHZ after 7 days of storage are as follows: Figure 4 As shown, the hydrodynamic diameter of the nano-regulator TPLHZ remained essentially unchanged after seven days in PBS, indicating that the nano-regulator has a stable and uniform particle size and good biological stability.

[0106] Test Example 2

[0107] The morphology of the prepared nano-modifier TK / PLT@Lip-HMME / Zeb was tested.

[0108] A small amount of TK / PLT@Lip-HMME / Zeb was diluted to 0.2 mg / mL with PBS. 20 μL of the suspension was repeatedly dropped onto a carbon-coated copper grid. After drying for 10 minutes, excess nanomaterials were absorbed with filter paper. The copper grid was then stained with 20 μL of 3% uranium acetate in the dark for 10 minutes. After drying, the grid was observed and photographed using a transmission electron microscope to analyze the morphology of the nanomaterials. The results are as follows: Figure 5 As shown.

[0109] Test Example 3

[0110] Co-location tests were performed on TK and PLT in the hybrid membrane.

[0111] First, TK and PLT obtained in Example 1 were sonicated for 5 min each. Then, PLT was stained with 1,1'-octadecyl-3,3,3',3'-tetramethylindole carbocyanine perchlorate (DiI), and TK was stained with 3,3'-octadecyloxycarbocyanine perchlorate (DiO). Next, PK and TLT were incubated at 37°C for 10 min at a 1:1 protein ratio. Finally, the co-localization of TK and PLT was observed using a fluorescence microscope. The results are as follows: Figure 6 As shown, TK and PLT can exhibit good co-positioning effect.

[0112] Test Example 4

[0113] The prepared nano-modifier TK / PLT@Lip-HMME / Zeb was subjected to a full-wavelength ultraviolet scan.

[0114] Successful loading of HMME and Zeb onto nanocarriers was determined using a UV spectrophotometer. Bare HMME, bare Zeb, physically mixed HMME+Zeb, and nanomodifier TK / PLT@Lip-HMME / Zeb (TPLHZ) were diluted and scanned in the wavelength range of 200 nm to 600 nm using a UV spectrophotometer.

[0115] The results are as follows Figure 7 As shown, the nano-modifier TK / PLT@Lip-HMME / Zeb exhibits significant UV absorption at both the maximum absorption wavelength of HMME (398 nm) and Zeb (398 nm), indicating the successful construction of the nanocomposite.

[0116] Test Example 5

[0117] The blood compatibility of the prepared nanomodifier TK / PLT@Lip-HMME / Zeb was studied.

[0118] First, a 2% fresh red blood cell suspension was prepared. After co-incubating the suspension with different concentrations of nanocarriers (TK / PLT@Lip, whose preparation method differs from Example 1 only in that the lipid nanocore is not loaded with HMME and Zeb, and its preparation is as described in Example 1) and nanomodifier TK / PLT@Lip-HMME / Zeb at 37°C for 1 h, the suspension was centrifuged, and the absorbance of the resulting supernatant was measured at 540 nm. PBS buffer was used as a negative control, and Triton X-100 (1%) was used as a positive control. The hemolysis index was calculated using the following formula:

[0119]

[0120] The results are as follows Figure 8 As shown, the concentration of nanoparticles and the lysis of erythrocytes generally exhibit a positive linear correlation. In the negative control tube, intact erythrocytes without hemolytic activity were observed in the supernatant, forming a firm, dark red precipitate at the bottom of the tube. According to ASTM E 2524-2008, the nanocarrier (TK / PLT@Lip) and nanoparticles (TK / PLT@Lip-HMME / Zeb) are in a relatively safe state in blood circulation, with hemolysis index within the normal range. The synthesized drug-loaded nanoparticles exhibit low hemolytic activity in blood, laying the foundation for the successful conduct of in vivo experiments.

[0121] Test Example 6

[0122] The antitumor activity of the prepared nanomodulator TK / PLT@Lip-HMME / Zeb was studied at the cellular level.

[0123] (1) Use the typical MTT reduction assay to assess cytotoxicity.

[0124] First, 4T1 cells or CT26 cells were seeded into 96-well plates. After cell adhesion, the cells were drugged. Different drug concentrations were set up as experimental groups, and the complete culture medium was set up as the control group. 4T1 cells or CT26 cells were incubated with HMME, Zeb, TK / PLT@Lip-HMME (the difference from the product in Example 1 is that it is loaded with only HMME and not Zeb, and the preparation method is the same as in Example 1), TK / PLT@Lip-Zeb (the difference from the product in Example 1 is that it is loaded with only Zeb and not HMME, and the preparation method is the same as in Example 1), and TK / PLT@Lip-HMME / Zeb (TPLHZ) for 12 h under different drug concentrations. Then, they were incubated with or without ultrasound irradiation (US) for another 12 h. Then, 100 μL of MTT solution (0.5 mg / mL) was added, and the cells were incubated with 5% carbon dioxide at 37°C in the dark for 4 h. Then, 100 μL of dimethyl sulfoxide (DMSO) was added, and after thorough shaking to dissolve, the absorbance of each well was measured at a wavelength of 570 nm using an ELISA reader. The cell viability of each experimental group was calculated to evaluate the toxicity of the constructed nanobody to normal cells and tumor cells. The experiment was repeated 3 times. (In each treatment group of this invention, the dosage of the corresponding drug was kept consistent. That is, the dosage of HMME in the TPLHZ group was the same as that in the TK / PLT@Lip-HMME group and the HMME group, and the dosage of Zeb in the TPLHZ group was the same as that in the TK / PLT@Lip-Zeb group and the Zeb group. Subsequent experiments were the same and will not be repeated.)

[0125] The experimental results of the 4T1 cell group are as follows: Figure 9 As shown, the experimental results of the CT26 cell group are as follows: Figure 10 As shown, the survival rates of cells treated with TK / PLT@Lip-HMME, TK / PLT@Lip-Zeb, and TPLHZ nanoparticles were lower than those treated with naked HMME and naked Zeb, indicating that nanoparticles can selectively accumulate in tumor cells, enhancing their anti-tumor effects. The number of cells in the ultrasound-treated groups was lower than in the untreated groups, because the ultrasound response is more conducive to the action of HMME, inducing immunogenic cell death in tumor cells. Furthermore, the survival rate of cells treated with TPLHZ was even lower than that of TK / PLT@Lip-HMME and TK / PLT@Lip-Zeb, because both HMME and Zeb simultaneously amplified the anti-tumor effects of the cells.

[0126] (2) Intracellular ROS levels were detected by staining with the fluorescent dye 2,7-dichlorodihydrofluorescein (DCFH-DA).

[0127] 4T1 cells (1×10) 5 Cells were seeded at 500 cells / well in 6-well plates and co-incubated with PBS, PBS (+US), free HMME+Zeb (+US), PLT@Lip-HMME / Zeb (+US), TK / PLT@Lip-HMME / Zeb, and TK / PLT@Lip-HMME / Zeb (+US), respectively. After 6 hours, the groups requiring ultrasound treatment were irradiated with ultrasound. After 4 hours, the culture medium was discarded, and the cells were washed with cold PBS. The cells were then incubated with 5 μL of DCFH-DA (10 mM) probe for 40 minutes, and the fluorescence intensity of the cells was observed under a fluorescence microscope.

[0128] The results are as follows Figure 11 As shown, 4T1 cells in the free HMME+Zeb(+US) group exhibited a slight increase in green fluorescence under ultrasound treatment, which is attributed to the generation of ROS by HMME sonodynamic therapy. 4T1 cells treated with PLT@Lip-HMME / Zeb(+US) and TK / PLT@Lip-HMME / Zeb showed stronger green fluorescence than the free HMME+Zeb(+US) group, possibly due to the efficient drug delivery capability of the nanocarriers. The significantly enhanced green fluorescence in cells treated with TK / PLT@Lip-HMME / Zeb(+US) indicates that catalase on the TK membrane effectively increases the oxygen content in the tumor microenvironment and enhances ROS generation under ultrasound irradiation.

[0129] Test Example 7

[0130] Animal studies were conducted on the antitumor activity of the prepared nanomodulator TK / PLT@Lip-HMME / Zeb.

[0131] (1) 4T1 cells (1×10 6 The tumor was resuspended in PBS and injected subcutaneously into the right hind limb of mice to construct a 4T1 subcutaneous tumor model.

[0132] (2) Seven days later, the mice were randomly divided into 6 groups, with an average tumor volume of 100 mm in each group. 3(n=5). Subsequently, mice were injected via tail vein with PBS (G1 group), PBS (+US) (G2 group), free HMME+Zeb (+US) (G3 group), PLT@Lip-HMME / Zeb (+US) (G4 group), TK / PLT@Lip-HMME / Zeb (G5 group), and TK / PLT@Lip-HMME / Zeb (+US) (G6 group) (HMME 8 mg / kg, Zeb 10 mg / kg). Drug treatment and ultrasound irradiation were performed every two days. The in vivo therapeutic effect of the drugs was observed by monitoring tumor volume and body weight in the mice.

[0133] (3) After treatment, the mice were euthanized by cervical dislocation. The tumor tissue was then dissected, and the tumor volume was calculated as length × width. 2 / 2(mm 3 ), where the length is the longest dimension and the width is the vertical dimension.

[0134] Tumor volume results for each group of mice are as follows: Figure 12 As shown, groups G4 and G5 exhibited moderate inhibitory effects, while group G6 significantly inhibited tumor growth, with a tumor inhibition rate of 52% that of the control group. This indicates that under ultrasound irradiation, the nanomodulator involved in this invention can maximally delay tumor growth.

[0135] The changes in body weight of mice in each group are as follows: Figure 13 As shown, the nano-regulators involved in this invention have good biosafety.

[0136] Test Example 8

[0137] Study on the enhanced antitumor effect of the prepared nanomodulator TK / PLT@Lip-HMME / Zeb combined with antiPD-1 antibody.

[0138] (1) 4T1 cells (1×10 6 The tumor was resuspended in PBS and injected subcutaneously into the right hind limb of mice to construct a 4T1 subcutaneous tumor model.

[0139] (2) Seven days later, the mice were randomly divided into four groups, with an average tumor volume of 100 mm in each group. 3(n=5). Subsequently, mice were administered PBS (G1 group), anti-PD-1 antibody (G2 group), TK / PLT@Lip-HMME / Zeb(+US) (G3 group), or TK / PLT@Lip-HMME / Zeb+anti-PD-1 antibody(+US) (G4 group) via tail vein injection or intraperitoneal injection (HMME 8 mg / kg, Zeb 10 mg / kg; anti-PD-1 antibody 5 mg / kg, intraperitoneal injection). Drug treatment and ultrasound irradiation were performed every two days. The in vivo therapeutic effect of the drugs was observed by monitoring tumor volume in the mice.

[0140] (3) After treatment, the mice were euthanized by cervical dislocation. The tumor tissue was then dissected, and the tumor volume was calculated as length × width. 2 / 2(mm 3 ), where the length is the longest dimension and the width is the vertical dimension.

[0141] The changes in body weight of mice in each group are as follows: Figure 14 As shown, groups G2 and G3 exhibited moderate inhibitory effects, while group G4 showed significant inhibitory effects, with a tumor inhibition rate of 69% compared to the control group. These results indicate that the combination therapy of the nanomodulator involved in this invention with anti-PD-1 antibody can effectively inhibit tumor growth.

[0142] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0143] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0144] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A biomimetic epigenetic nanoregulator based on ultrasonic response, characterized in that, The ultrasound-responsive biomimetic epigenetic nanomodulator comprises: a lipid nanocore and a hybrid biomembrane fused thereto; The lipid nanocore comprises: a liposome and a sonosensitive agent loaded in its lipid phase and a DNA methyltransferase inhibitor loaded in its aqueous phase; The hybrid biomembrane is a platelet membrane and a thylakoid membrane fused with it; The sonosensitive agent is hematoporphyrin monomethyl ether, and the DNA methyltransferase inhibitor is zablaline.

2. The biomimetic epigenetic nanoregulator based on ultrasonic response according to claim 1, characterized in that, The raw materials for preparing the liposomes include phospholipids, cholesterol, and DSPE-PEG.

3. The ultrasonic-responsive biomimetic epigenetic nanoregulator according to claim 2, characterized in that, The phospholipids include soybean lecithin.

4. The biomimetic epigenetic nanoregulator based on ultrasonic response according to claim 2, characterized in that, The number-average molecular weight of PEG in the DSPE-PEG is 1000-4000.

5. The biomimetic epigenetic nanoregulator based on ultrasonic response according to claim 1, characterized in that, The mass ratio of the liposome to the sound-sensing agent is (1-10):(0.1-1).

6. The biomimetic epigenetic nanoregulator based on ultrasonic response according to claim 1, characterized in that, The mass ratio of the sound sensitizer to the DNA methyltransferase inhibitor is (1-10):(1.5-15).

7. The biomimetic epigenetic nanoregulator based on ultrasonic response according to claim 1, characterized in that, The protein concentration ratio of the platelet membrane to the thylakoid membrane is (1-5):(1-5).

8. The biomimetic epigenetic nanoregulator based on ultrasonic response according to claim 1, characterized in that, The mass ratio of the lipid nanocore to the hybrid biomembrane is (10-100):(1-10).

9. The method for preparing the ultrasonic-responsive biomimetic epigenetic nanoregulator according to any one of claims 1-8, characterized in that, The preparation method includes: (1) A lipid film is obtained by rotary evaporation of an organic mixture containing liposome raw materials and a sound-sensing agent; (2) The DNA methyltransferase inhibitor solution was mixed with the lipid film and hydrated to obtain the lipid nanocore; (3) The lipid nanocore was mixed with the hybrid biomembrane and co-incubated to obtain the ultrasound-responsive biomimetic epigenetic nanoregulator.

10. The method for preparing the ultrasonic-responsive biomimetic epigenetic nanoregulator according to claim 9, characterized in that, The method for preparing the hybrid biomembrane includes: mixing and co-incubating platelet membrane and thylakoid membrane.

11. The method for preparing the ultrasonic-responsive biomimetic epigenetic nanoregulator according to claim 10, characterized in that, The co-incubation was carried out at 30-45℃ for 5-20 minutes.

12. The method for preparing the ultrasonic-responsive biomimetic epigenetic nanoregulator according to claim 10, characterized in that, Ultrasonic assisted treatment is also added during the co-incubation process.

13. The method for preparing the ultrasonic-responsive biomimetic epigenetic nanoregulator according to claim 10, characterized in that, The platelet membrane is an autologous platelet membrane.

14. The method for preparing the ultrasonic-responsive biomimetic epigenetic nanoregulator according to claim 10, characterized in that, The platelet membrane is prepared by differential centrifugation, specifically including: (S1) Centrifuge blood at 80-150 g for 10-40 min; (S2) Take the supernatant and centrifuge at 80-150 g for 10-40 min; (S3) Take the supernatant and centrifuge at 700-1000 g for 10-40 min to collect platelets.

15. The method for preparing the ultrasonic-responsive biomimetic epigenetic nanoregulator according to claim 10, characterized in that, The method for preparing the thylakoid membrane is differential centrifugation, specifically including: (T1) After homogenizing the spinach, filter it and centrifuge the filtrate at 6000-8000 rpm for 5-20 min; (T2) Resuspend the sediment and centrifuge at 10000-14000 g for 20-40 min; (T3) Resuspend the sediment and centrifuge at 14000-20000 g for 10-25 min to collect the thylakoid membrane.

16. The method for preparing the ultrasonic-responsive biomimetic epigenetic nanoregulator according to claim 9, characterized in that, The rotary evaporation in step (1) is carried out at 45-60℃ for 30-180 min.

17. The method for preparing the ultrasonic-responsive biomimetic epigenetic nanoregulator according to claim 9, characterized in that, The hydration in step (2) is carried out at 20-25°C.

18. The method for preparing the ultrasonic-responsive biomimetic epigenetic nanoregulator according to claim 9, characterized in that, The co-incubation in step (3) is carried out at 30-45℃ for 5-20 min.

19. The application of the ultrasound-responsive biomimetic epigenetic nanomodulator according to any one of claims 1-8 in the preparation of antitumor drugs.

20. The application according to claim 19, characterized in that, The tumor is a PD-1 resistant tumor.

21. The use of the ultrasound-responsive biomimetic epigenetic nanomodulator according to any one of claims 1-8 in the preparation of a formulation that upregulates the expression of MHC-I on the surface of tumor cells.

22. A combination pharmaceutical composition for antitumor use, characterized in that, The combined pharmaceutical composition comprises the ultrasound-responsive biomimetic epigenetic nanomodulator and the anti-PD-1 antibody as described in any one of claims 1-8.

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