MXene compound coated with cancer cell membrane and loaded with STING agonist as well as preparation method and application of MXene compound

Abstract

The invention discloses a cancer cell membrane coated STING agonist loaded MXene compound and a preparation method and application thereof, and belongs to the technical field of biomedical materials.The preparation method comprises the steps that Ti3AlC2 and LiF are mixed, a concentrated hydrochloric acid solution is added, a reaction is conducted for 24-72 h at the temperature of 25-50 DEG C, and then centrifugal washing is conducted to obtain a black precipitate; adding the black precipitate into water, carrying out ultrasonic treatment under the protection of nitrogen to obtain a colloidal solution, and then centrifugally collecting supernate to obtain a d-MXene solution; the preparation method comprises the following steps: mixing a d-MXene solution and an STING agonist solution, stirring at room temperature, reacting in a dark place, adding a micromolecule enhancer containing an amide group into the mixture during the reaction to obtain an STING agonist-loaded MXene compound, and further coating by using a liver cancer cell membrane to obtain a cancer cell membrane-coated STING agonist-loaded MXene compound. The compound has a wide application prospect in the aspect of hepatocellular carcinoma treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to an MXene complex loaded with STING agonist encapsulated in a cancer cell membrane, its preparation method, and its application. Background Technology

[0002] Liver cancer is one of the most common malignant tumors of the digestive system. It is highly insidious, progresses rapidly, and is difficult to cure if it develops to an advanced stage. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Persistent liver disease, alcohol intake, chemical exposure, liver damage, and obesity can all induce HCC. For early and late-stage HCC, clinicians use techniques such as irreversible electroporation (IRE), photothermal therapy (PTT), photodynamic therapy (PDT), sonodynamic therapy (SDT), magnetothermal therapy (MHT), and radiofrequency ablation (RFA), combined with chemotherapy drugs such as doxorubicin and cisplatin, to implement comprehensive treatment to reduce the risk of recurrence. However, traditional chemotherapy drugs often have problems with poor targeting and dose-limited toxicity.

[0003] Nanomedicines, with their unique physicochemical properties (such as small size, high specific surface area, and ability to be functionalized) and precise ability to regulate the tumor microenvironment, have shown great potential in the treatment of hepatocellular carcinoma and have become a research hotspot in the field of tumor nanomedicine. However, nanomaterials generally suffer from limited water solubility. Two-dimensional MXene materials are hydrophilic and are represented by the general formula Mn+1XnTx, where M represents an early transition metal, X represents C or N, and Tx is a surface group, usually -OH, -O, -F, and -Cl. In cancer treatment, MXene shows promising application prospects. For example, Chinese patent document CN114804106A discloses a vanadium-titanium carbide MXene material, its preparation method, and its applications. This vanadium-titanium carbide MXene material exhibits broad light absorption in the near-infrared region, with high absorption rates in the 1100-2500 nm range, demonstrating potential for application in the near-infrared long-wavelength region. Furthermore, it exhibits good biocompatibility, is non-toxic to normal cells, and possesses tumor-targeting properties. Under near-infrared light excitation, it can generate a strong thermal effect to eliminate tumors, and can be combined with photothermal and chemical dynamics. In the field of tumor treatment, Chinese patent document CN115531537A discloses an MXene@cuttlefish juice melanin complex with photothermal synergistic antibacterial properties, its preparation method, and its applications. This invention uses natural cuttlefish juice melanin as a template and uses a simple stirring method to encapsulate or cover monolayer MXene onto cuttlefish juice melanin through hydrogen bonding, thereby constructing an MXene@cuttlefish juice melanin complex with a core-shell-like assembly and good dispersibility in one step. The MXene@cuttlefish juice melanin complex has broad application prospects in the field of tumor suppression.

[0004] However, the MXene materials or MXene material complexes mentioned above do not have a specific effect on hepatocellular carcinoma. Therefore, it is necessary to develop a novel MXene complex for the treatment of hepatocellular carcinoma. Summary of the Invention

[0005] This invention provides a method for preparing a cancer cell membrane-encapsulated MXene complex loaded with a STING agonist. The method is simple and efficient, and the resulting cancer cell membrane-encapsulated MXene complex loaded with a STING agonist exhibits good water solubility, high cellular uptake efficiency, and good anti-tumor effect.

[0006] The specific technical solution adopted is as follows:

[0007] A method for preparing an MXene complex loaded with a STING agonist and encapsulated in a cancer cell membrane, comprising the following steps:

[0008] (1) Mix Ti3AlC2 with LiF, add concentrated hydrochloric acid solution, react at 25-50℃ for 24-72h, centrifuge and wash until the pH of the supernatant is ≥6, and obtain a black precipitate;

[0009] (2) The black precipitate was added to water and ultrasonically treated under nitrogen protection to obtain a colloidal solution. The colloidal solution was centrifuged and the supernatant was collected to obtain a d-MXene solution.

[0010] (3) Mix the d-MXene solution with the STING agonist solution, stir and react at room temperature in the dark, and add a small molecule enhancer containing an amide group during the reaction to obtain the MXene complex loaded with the STING agonist.

[0011] (4) The MXene complex loaded with STING agonist was obtained by encapsulating the liver cancer cell membrane with the MXene complex loaded with STING agonist.

[0012] This invention utilizes MXene to load the STING agonist. The loading process needs to be carried out under light-protected conditions, as exposure to light induces photo-oxidation and aggregation. The MXene complex containing the STING agonist, encapsulated in cancer cell membranes prepared in this invention, generates significant local heat (40-55°C) and reactive oxygen species (ROS) even at low concentrations (1-5 μg / mL in culture medium) under NIR-I (808 nm) laser irradiation. This leads to the controlled release of the STING agonist from the complex system, thereby activating the STING pathway and initiating downstream immune responses. Simultaneously, the ROS and photothermal effects cause MXene to fragment into smaller nanosheets, enhancing its intracellular and organelle penetration. Intracellularly, the Ti... 2 + / Ti3 + and F- species impair mitochondrial membrane potential and disrupt the electron transport chain (ETC), leading to energy depletion and cell death. Furthermore, the MXene complex loaded with the STING agonist encapsulated in the cancer cell membrane can downregulate the expression of vascular endothelial growth factor (VEGF), thereby inhibiting angiogenesis. In summary, the liver cancer cell membrane-encapsulated MXene complex loaded with the STING agonist prepared in this invention exhibits a synergistic therapeutic effect on cancer cells by combining immune activation, oxidative stress, photothermal ablation, mitochondrial dysfunction, and anti-angiogenic activities.

[0013] Simultaneously, in the preparation process of the STING agonist-loaded MXene complex, this invention also incorporates a small molecule enhancer containing an amide group. The introduction of this small molecule enhancer improves the stability, dispersibility, and intracellular delivery efficiency of the complex. The amide-containing small molecule enhancer enhances the colloidal stability of MXene by inhibiting oxidation and aggregation. Furthermore, due to their high polarity and solubility gradient, these molecules promote the effective loading of the STING agonist onto MXene by improving molecular interactions and surface wettability, thereby increasing the loading rate of the STING agonist. When exposed to near-infrared (NIR-I) laser light, the small molecule enhancer facilitates the controlled cleavage of MXene, thereby promoting enhanced subcellular penetration. The corresponding small molecule enhancer also increases cellular uptake and membrane permeability.

[0014] Preferably, in step (1), Ti3AlC2 and LiF are mixed in equal mass ratio and then reacted with pre-cooled concentrated hydrochloric acid solution.

[0015] Preferably, in step (2), the patient is ultrasonically treated at 4-15℃ for 0.5-6 hours under nitrogen protection.

[0016] Specifically, the STING agonist is diABZI STING agonist (Compound 3), with the molecular formula C. 42 H 51 N 13 O7, the structural formula is shown below:

[0017]

[0018] Preferably, in step (3), the concentration of the d-MXene solution is 50-1000 μg / mL, the concentration of the STING agonist solution is 50-200 μg / mL, and the volume ratio of the d-MXene solution to the STING agonist solution is 1:0.001-0.8.

[0019] More preferably, in step (3), the reaction is carried out at room temperature with stirring and in the dark for 3-24 hours.

[0020] Optionally, the amide-containing small molecule enhancer is selected from 2-pyrrolidone, 1,3-dimethyl-2-imidazolinone, succinimide, N-methyl-2-pyrrolidone, phthalimide, N-formylpyrrolidine, caprolactam, or N-ethyl-2-pyrrolidone, and the mass ratio of d-MXene to the amide-containing small molecule enhancer is 1:0.001-1. The amide-containing small molecule enhancer can increase cellular uptake efficiency several times and shorten uptake time.

[0021] Specifically, in step (4), the liver cancer cell membrane is selected from Hepa1-6 cell membranes, and the liver cancer cell membrane is encapsulated with an MXene complex loaded with STING agonist by co-extrusion or ultrasonic method.

[0022] The present invention also provides a method for preparing the cancer cell membrane-encapsulated MXene complex loaded with STING agonist, and the resulting cancer cell membrane-encapsulated MXene complex loaded with STING agonist.

[0023] The present invention also provides the application of the aforementioned cancer cell membrane-encapsulated MXene complex loaded with the STING agonist in the preparation of hepatocellular carcinoma therapeutic drugs.

[0024] The present invention also provides a hepatocellular carcinoma treatment drug comprising the aforementioned cancer cell membrane-encapsulated MXene complex loaded with a STING agonist.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) This invention utilizes d-MXene, STING agonist, small molecule enhancer containing amide group and liver cancer cell membrane to prepare a surface-engineered nanostructure. STING agonist has the dual functions of ligand and bioactive molecule, without the need for the introduction of other polymer carriers, avoiding unnecessary volume expansion and complexity. The preparation method is simple and efficient.

[0027] (2) The cancer cell membrane-encapsulated MXene complex loaded with STING agonist prepared in this invention, d-MXene, STING agonist, small molecule enhancer containing amide group and liver cancer cell membrane work synergistically to produce multimodal therapeutic effects on cancer cells by combining immune activation, oxidative stress, photothermal ablation, mitochondrial dysfunction and anti-angiogenic activity, and has broad application prospects in the field of hepatocellular carcinoma treatment. Attached Figure Description

[0028] Figure 1 The images show scanning electron microscope images of the MAX phase and the exfoliated MXene, where (a) is the MAX phase, and (b) and (c) are the exfoliated monolayer MXene.

[0029] Figure 2 XRD patterns of Ti3AlC2 and d-Ti3C2Tx(d-MXene).

[0030] Figure 3 (a) shows the attenuated total reflectance Fourier transform infrared (ATR-FTIR) characterization results of the MXene complex loaded with STING agonist, and (b) shows the analysis results of gas chromatography-mass spectrometry (GC-MS).

[0031] Figure 4 Zeta potential test results for MXene complex loaded with STING agonist and control substance encapsulated in cancer cell membrane.

[0032] Figure 5 The results of application characterization of the STING agonist-loaded MXene complex (MSCM) encapsulated in cancer cell membranes are shown in the following figures: (a) shows the relationship between MSCM dose and temperature in DMEM+FBS medium; (b) shows the results of real-time quantitative polymerase chain reaction (RT-qPCR) of STING agonist alone; and (c) shows the results of real-time quantitative polymerase chain reaction (RT-qPCR) of MSCM. Detailed Implementation

[0033] To make the objectives, features, and advantages of this invention more apparent and understandable, a detailed description is provided below through specific embodiments. Many specific details are set forth in the following description to provide a thorough understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below. Technical features in various embodiments of the invention can be combined appropriately without mutual conflict.

[0034] In the following examples, the STING agonist used is the commercially available product diABZI STING agonist (Compound3), with the following structural formula:

[0035]

[0036] Example 1

[0037] Ti3AlC2 and LiF were mixed at a mass ratio of 1:1, and 20 mL of pre-cooled 9M HCl solution was added (the reaction is exothermic and requires an ice bath). After reacting with magnetic stirring at 35°C for 24 hours, the mixture was centrifuged at 3500 rpm for 5 minutes and washed until the pH of the supernatant was ≥6, resulting in a black precipitate. The black precipitate was added to 10 mL of deionized water and sonicated at a temperature below 15°C under nitrogen protection for 6 hours to obtain a colloidal solution. The colloidal solution was centrifuged at 4000 rpm for 1 hour, and the supernatant was collected to obtain a d-MXene solution.

[0038] A 50 μg / mL d-MXene solution (solvent: H2O / PBS) and a 200 μg / mL STING agonist solution (solvent: H2O / PBS) were mixed at a volume ratio of 1:0.8 and reacted at room temperature in the dark for 12 h. During the reaction, N-methyl-2-pyrrolidone was added to the mixture, with a mass ratio of d-MXene to N-methyl-2-pyrrolidone of 1:0.8, to obtain an MXene complex loaded with STING agonist.

[0039] Hepa1-6 cells were cultured in a medium containing a 9:1 volume ratio of DMEM and FBS. After passage, the cells were thoroughly washed with PBS, transferred to a hypotonic mixture, and incubated on ice for 1 hour. After sonication, the cells were centrifuged initially (3500 rpm / 5 min) to collect the supernatant, and then centrifuged again (12000 rpm / 10 min) to collect the precipitate. The resulting solid was washed with pure water and resuspended for later use (stored at -80℃) to obtain the Hepa1-6 cell membrane (CCM). The CCM solution was mixed with a solution of MXene complex loaded with STING agonist, and the CCM was coated with the STING agonist-loaded MXene complex using co-extrusion or sonication to obtain the cancer cell membrane-coated MXene complex loaded with STING agonist.

[0040] Example 2

[0041] The only difference between this embodiment and Example 1 is that 1000 μg / mL of d-MXene solution (solvent: H2O / PBS) and 200 μg / mL of STING agonist solution (solvent: H2O / PBS) were mixed at a volume ratio of 1:0.087 to prepare the MXene complex loaded with STING agonist encapsulated in the cancer cell membrane.

[0042] Sample Analysis

[0043] Figure 1The images show scanning electron microscope (SEM) images of the MAX phase and the exfoliated MXene. In the image, a represents the MAX phase, and b and c represent the exfoliated monolayer MXene. It can be seen that the layered structure of the MAX phase is composed of alternating stacked Ti-C(MX) layers and Al(A) layers. The exfoliated monolayer MXene exhibits edge curling, which is mainly attributed to the effect of interlayer hydrogen bonds. The structure is intact and without defects or with only a few breaks, indicating that MXene has good electrical conductivity, which in turn suggests that d-MXene has high thermal performance.

[0044] Figure 2 The XRD patterns of Ti3AlC2 and d-Ti3C2Tx (d-MXene) are shown. The sharp peaks in the XRD patterns indicate that both Ti3AlC2 and d-Ti3C2Tx possess crystalline properties. The broader characteristic peaks of d-Ti3C2Tx indicate the formation of a monolayer structure. The missing 38.82° peak in d-Ti3C2Tx corresponds to Al, and the (002) crystal plane peak shifts from 9.54° to 7.24°. The appearance of the (0012) crystal plane peak further confirms the ordered structure of d-MXene. Furthermore, the UV-Vis absorption spectroscopy of d-MXene shows a high absorbance at 775 nm, making it more suitable for the NIR I window. XPS analysis indicates that the STING agonist binds to d-MXene through hydrogen bonds or dipole-dipole interactions, and the d-MXene material conforms to the general formula of d-Ti3C2T(OH,F).

[0045] Figure 3 (a) shows the attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) characterization results of the MXene complex loaded with STING agonist, and (b) shows the analysis results of gas chromatography-mass spectrometry (GC-MS). Together, they verify the successful loading of STING molecules on d-MXene materials.

[0046] Figure 4 As shown in the Zeta potential test results, the MXene complex (MSCM) loaded with STING agonist encapsulated in the cancer cell membrane of Example 1 exhibits significant negative charge.

[0047] Figure 5The results in (a)-(c) demonstrate that the cancer cell membrane-encapsulated STING agonist-loaded MXene complex (MSCM) formulation of Example 1 exhibited significant photothermal activity after 3 minutes of NIR-I (808 nm) laser irradiation, with local temperatures ranging from 42°C to above 55°C. Notably, the biological activity of cells internalized with MSCM and subsequently exposed to NIR-I irradiation was completely inhibited. RT-qPCR results showed no detectable expression of key genetic markers, indicating effective cell ablation. This result was not observed in control samples treated with 0.001-1 μg / mL of free STING agonist, which showed limited or no gene regulation and minimal thermal effect. The cancer cell membrane-encapsulated STING agonist-loaded MXene complex MSCM prepared in Example 1 showed a significant effect. These results indicate that when the MSCM system is activated by photothermal stimulation, it induces irreversible cell shutdown through a combination of intracellular heat generation, ROS generation, and mechanical degradation, ultimately leading to loss of RNA integrity and complete inhibition of transcriptional activity, thereby exerting a therapeutic effect.

[0048] In summary, this invention has developed an MXene complex loaded with the STING agonist and encapsulated within a cancer cell membrane. This complex achieves stable loading of the STING agonist and utilizes the cancer cell membrane for surface camouflage. Furthermore, experiments have demonstrated that the combined application of irreversible electroporation (IRE) and near-infrared ionotropic laser (NIR-I) significantly enhances the anti-tumor effect in an in vitro model, and this combined treatment strategy reduces the dosage of the MXene complex to 1 / 40th of the conventional dose. Studies show that the MXene complex loaded with the STING agonist and encapsulated within the cancer cell membrane developed in this invention can efficiently kill cancer cells within the treatment window (photothermal effect) of irreversible electroporation (IRE) and NIR-I, providing a comprehensive treatment option for hepatocellular carcinoma (HCC).

[0049] The MSCM system independently reduces cellular biochemical activity by activating the STING pathway and other molecular interactions. Upon further exposure to photothermal stimulation, MSCM induces irreversible cell inactivation through synergistic mechanisms, including local intracellular heat generation, reactive oxygen species (ROS) production, and structural degradation. These combined effects lead to significant cellular damage, resulting in loss of viability and disruption of functional integrity, which may be beneficial for the treatment of hepatocellular carcinoma.

[0050] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an MXene complex loaded with a STING agonist and encapsulated in a cancer cell membrane, characterized in that, Includes the following steps: (1) Mix Ti3AlC2 with LiF, add concentrated hydrochloric acid solution, react at 25-50℃ for 24-72h, centrifuge and wash until the pH of the supernatant is ≥6, and obtain a black precipitate; (2) The black precipitate was added to water and ultrasonically treated under nitrogen protection to obtain a colloidal solution. The colloidal solution was centrifuged and the supernatant was collected to obtain a d-MXene solution. (3) Mix the d-MXene solution with the STING agonist solution, stir and react at room temperature in the dark, and add a small molecule enhancer containing an amide group during the reaction to obtain the MXene complex loaded with the STING agonist. (4) The MXene complex loaded with STING agonist was obtained by encapsulating the liver cancer cell membrane with the MXene complex loaded with STING agonist.

2. The method for preparing the MXene complex loaded with the STING agonist and encapsulated in the cancer cell membrane according to claim 1, characterized in that, Ti3AlC2 and LiF were mixed in equal mass ratio and then reacted with a pre-cooled concentrated hydrochloric acid solution.

3. The method for preparing the MXene complex loaded with the STING agonist and encapsulated in the cancer cell membrane according to claim 1, characterized in that, In step (2), the mixture is ultrasonically treated at 4-15℃ for 0.5-6 hours under nitrogen protection.

4. The method for preparing the MXene complex loaded with the STING agonist and encapsulated in the cancer cell membrane according to claim 1, characterized in that, The structural formula of the STING agonist is as follows:

5. The method for preparing the MXene complex loaded with the STING agonist and encapsulated in the cancer cell membrane according to claim 1, characterized in that, In step (3), the concentration of d-MXene solution is 50-1000 μg / mL, the concentration of STING agonist solution is 50-200 μg / mL, and the volume ratio of d-MXene solution to STING agonist solution is 1:0.001-0.

8.

6. The method for preparing the MXene complex loaded with the STING agonist and encapsulated in the cancer cell membrane according to claim 1, characterized in that, The amide-containing small molecule reinforcing agent is selected from 2-pyrrolidone, 1,3-dimethyl-2-imidazolinone, succinimide, N-methyl-2-pyrrolidone, phthalimide, N-formylpyrrolidine, caprolactam, or N-ethyl-2-pyrrolidone, and the mass ratio of d-MXene to the amide-containing small molecule reinforcing agent is 1:0.001-1.

7. The method for preparing the MXene complex loaded with the STING agonist and encapsulated in the cancer cell membrane according to claim 1, characterized in that, In step (4), the liver cancer cell membrane is selected from Hepa1-6 cell membranes, and the liver cancer cell membrane is encapsulated with an MXene complex loaded with STING agonist by co-extrusion or ultrasound.

8. The cancer cell membrane-encapsulated MXene complex loaded with STING agonist prepared by the method of preparing the cancer cell membrane-encapsulated MXene complex loaded with STING agonist according to any one of claims 1-7.

9. The use of the MXene complex loaded with the STING agonist, encapsulated in the cancer cell membrane according to claim 8, in the preparation of a therapeutic agent for hepatocellular carcinoma.

10. A drug for treating hepatocellular carcinoma, characterized in that, The MXene complex containing a STING agonist encapsulated in a cancer cell membrane as described in claim 8.

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