Hybrid bionic nano delivery system and preparation method thereof

Abstract

The invention provides a hybrid bionic nano delivery system and a preparation method thereof. The hybrid bionic nano delivery system is composed of a carbon dot doped nitrogen carbide-based nanoparticle core and a cell membrane group wrapping the nitrogen carbide-based nanoparticle core. The invention constructs a hybrid bionic nano delivery system, which has good biocompatibility and biological metabolism, is beneficial to application in the field of biomedicine, and can realize low-temperature photothermal therapy of hepatic fibrosis. The carbon nitride-based nano bionic system constructed by the invention is mainly synthesized by high-temperature calcination, ultrasonic treatment and hybrid cell membrane wrapping. The preparation method disclosed by the invention is simple, has good repeatability and has a relatively good industrial production prospect.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to a hybrid biomimetic nanodelivery system and its preparation method. Background Technology

[0002] In recent years, photothermal therapy has made significant progress in the treatment of various diseases due to its controllability, minimally invasive nature, and lack of drug resistance. Furthermore, nanomaterials such as carbon-based materials and metal oxides have also been reported to have good photothermal therapeutic effects. However, photothermal therapy for liver fibrosis typically requires local temperatures exceeding 70 °C, which often leads to inflammatory responses and damage to surrounding healthy liver tissue. In addition, multifunctional photothermal therapy systems are often rapidly cleared by the immune system, and their insufficient targeting specificity limits their efficacy.

[0003] Carbon nitride (also known as CN or C3N4) is a photocatalytic material that has attracted widespread attention in the biomedical field due to its excellent biocompatibility and unique structure. However, its application in biomedicine still faces the following challenges: First, as a photothermal therapy material, it is easily recognized and cleared by the immune system, and due to its insufficient targeting, it is difficult to reach the treatment site; second, its high stability in vivo makes it difficult to metabolize and easily accumulates in the body. Notably, a low-temperature photothermal therapy nanodelivery system with targeted liver fibrosis tissue has not yet been developed. Summary of the Invention

[0004] This application provides a hybrid biomimetic nanodelivery system and its preparation method to solve the above-mentioned technical problems.

[0005] To solve the above-mentioned technical problems, one technical solution adopted in this application is: a hybrid biomimetic nanodelivery system, which is composed of a carbon dot-doped nitrogen-based carbon nanoparticle core and a cell membrane assembly that encapsulates the nitrogen-based carbon nanoparticle core.

[0006] Furthermore, the cell membrane group is composed of RAW264.7 cell membrane and aHSCs cell membrane in a ratio of 1:0.5 to 1.

[0007] Furthermore, the diameter of the nitrogen-based carbon nanoparticles is approximately 100-200 nm.

[0008] Furthermore, the hybrid biomimetic nanodelivery system possesses CAT enzyme activity to improve hypoxic conditions in liver fibrotic tissue; the hybrid biomimetic nanodelivery system can achieve photothermal therapy for liver fibrosis at low temperatures.

[0009] Another technical solution adopted in this application is: a method for preparing a hybrid biomimetic nanodelivery system.

[0010] S1. The carbon dot precursor and the nitrogen-containing precursor are mixed and dried in an oven, and then calcined at high temperature under nitrogen protection to obtain carbon dot-doped carbon nitride (CCNs).

[0011] S2. Disperse carbon doped carbon nitride (CCN) powder in water and sonicate it to obtain a mixed solution;

[0012] S3. The CCNs nanomaterials in the mixed solution were combined with the extracted RAW264.7 and aHSCs cell membrane proteins to obtain the HMCCNs hybrid biomimetic nanodelivery system.

[0013] Furthermore, in step S1, the nitrogen-containing precursor is melamine, and the carbon dot precursor is carbon quantum dots; the ratio of melamine to carbon quantum dots is 1:(0.05-0.1); the calcination temperature is 400-700 ℃; the calcination time is 4-8 h; and the heating rate is 3-10 ℃ / min.

[0014] Furthermore, in step S2, the ultrasonic treatment time is 2-5 hours and the ultrasonic power is 300W.

[0015] Furthermore, in step S3, the ratio of RAW264.7 to aHSCs cell membranes is 1:(0.5-1), the ratio of cell membrane complex to CCNs is 1:(0.5-10), and the sonication time is 2-5 h.

[0016] The beneficial effects of this application are:

[0017] (1) This invention constructs a hybrid biomimetic nanodelivery system with good biocompatibility and biometabolic properties, which is beneficial for application in the biomedical field and can realize low-temperature photothermal therapy for liver fibrosis. The nitride-based carbon nanomimetic system constructed in this application is mainly synthesized by high-temperature calcination, ultrasonic treatment, and hybrid cell membrane encapsulation. The preparation method of this invention is simple, has good reproducibility, and has good prospects for industrial production.

[0018] (2) The hybrid biomimetic nanodelivery system CCNs of this application can effectively bind to the cell membrane and form stable nanoparticles.

[0019] (3) The cell membrane encapsulation technology of the hybrid biomimetic nanodelivery system CCNs provided in this application enhances its targeting of liver fibrosis tissue and has good biocompatibility and therapeutic ability.

[0020] (4) The nitride-based hybrid biomimetic nanodelivery system provided in this application has CAT enzyme-like activity, which can improve the hypoxic conditions of liver fibrosis tissue and achieve low-temperature photothermal therapy on liver fibrosis tissue. Attached Figure Description

[0021] Figure 1 The X-ray diffraction (XRD) pattern of carbon dopant carbon nitride-based CCNs in Example 1 of this application;

[0022] Figure 2 This is an elemental mapping diagram of the hybrid biomimetic nanodelivery system HMCCNs in Embodiment 1 of this application;

[0023] Figure 3 Transmission electron microscopy (TEM) images of the hybrid biomimetic nanodelivery system in Embodiment 1 of this application;

[0024] Figure 4 The image shows the Coomassie Brilliant Blue results of the hybrid biomimetic nanodelivery system HMCCNs and RAW264.7, aHSCs cell membrane proteins in Example 2 of this application.

[0025] Figure 5 The temperature variation of the hybrid biomimetic nanodelivery system HCCNs in Embodiment 3 of this application under different power lasers;

[0026] Figure 6 Evaluation of the in vitro H2O2 decomposition activity and CAT enzyme catalytic performance of carbon doped carbon nitride-based CCNs in Example 4 of this application.

[0027] Figure 7 This is a CLSM imaging diagram of the CAT enzyme activity of the hybrid biomimetic nanodelivery system HMCCNs in aHSCs cells, as described in Example 5 of this application.

[0028] Figure 8 This is a live imaging image of the hybrid biomimetic nanodelivery system HMCCNs in Example 6 of this application. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments.

[0030] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification.

[0031] This application provides a hybrid biomimetic nanodelivery system, which consists of a carbon-doped nitrogen-based carbon nanoparticle core and a cell membrane encapsulating the nitrogen-based carbon nanoparticle core. The cell membrane assembly is composed of RAW264.7 cell membranes and aHSCs cell membranes in a ratio of 1:0.5 to 1. The hybrid biomimetic nanodelivery system possesses CAT enzyme activity, which can improve the hypoxic conditions of liver fibrosis tissue; the hybrid biomimetic nanodelivery system can achieve photothermal therapy for liver fibrosis at low temperatures. The nitrogen-based carbon nanoparticle core has a diameter of approximately 100-200 nm.

[0032] Among them, the biomimetic nanozyme uses carbon doped carbon nitride as the core and constructs it into a hybrid biomimetic nanodelivery system with low-temperature therapeutic capabilities; the hybrid biomimetic nanodelivery system can avoid immune clearance by the body when combined with the RAW264.7 cell membrane; the hybrid biomimetic nanodelivery system can achieve targeted aggregation of liver fibrosis tissue when combined with the aHSCS cell membrane.

[0033] Another technical solution adopted in this application is: a method for preparing a hybrid biomimetic nanodelivery system.

[0034] S1. The carbon dot precursor and the nitrogen-containing precursor are mixed and dried in an oven, and then calcined at high temperature under nitrogen protection to obtain carbon dot-doped carbon nitride (CCNs); wherein the nitrogen-containing precursor is melamine and the carbon dot precursor is carbon quantum dots; the ratio of melamine to carbon quantum dots is 1:(0.05-0.1); the calcination temperature is 400-700 ℃; the calcination time is 4-8 h; and the heating rate is 3-10 ℃ / min.

[0035] S2. Disperse carbon doped carbon nitride (CCN) powder in water and sonicate it for 2-5 hours at a power of 300W to obtain a mixed solution.

[0036] S3. The CCNs nanomaterials in the mixed solution were combined with the extracted RAW264.7 and aHSCs cell membrane proteins to obtain an HMCCNs hybrid biomimetic nanodelivery system. The ratio of RAW264.7 to aHSCs cell membranes was 1:(0.5-1), the ratio of cell membrane complex to CCNs was 1:(0.5-10), and the sonication time was 2-5 h.

[0037] Example 1

[0038] A novel hybrid biomimetic nanodelivery system for cryogenic photothermal therapy of liver fibrosis is proposed. The system uses carbon dot-doped carbon nitride nanoparticles as the core and further modifies the cell membranes of RAW264.7 and aHSCs to construct a nitrogen-based hybrid biomimetic nanodelivery system.

[0039] The preparation method of the hybrid biomimetic nanodelivery system for cryogenic photothermal therapy of liver fibrosis described in this embodiment includes the following steps:

[0040] (1) Weigh melamine and carbon dots in a mass ratio of 1:(0.05-0.1), mix them evenly, and dry them in an oven at 60-70℃. Calcine the resulting powder in a tube furnace at 400-700℃ for 4-8 hours, with a heating rate of 3-10℃ / min. The final calcined product is CCNs.

[0041] (2) The obtained CCNs powder was ultrasonically treated for 2-5 h with an ultrasonic power of 300W to ensure complete dispersion.

[0042] (3) Mix RAW264.7 and aHSCs cell membrane proteins at a ratio of 1:(0.5-1), and then sonicate the cell membrane protein complex with CCNs at a ratio of 1:(0.5-10) for 2-5 h. The nitrogen-based hybrid biomimetic nanodelivery system is thus obtained.

[0043] Please see Figure 1 ,from Figure 1 It can be seen that the present invention successfully doped carbon dots into carbon nitride, and the crystal form of the nanosheet did not change significantly.

[0044] Please see Figure 2 and Figure 3 ,from Figure 2 and Figure 3 It can be seen that the presence of phosphorus indicates that the cell membranes of RAW264.7 and aHSCs were also successfully coated with nanoenzymes. The above characterization shows that HMCCNs were successfully prepared, and after being introduced into the cell membrane to coat the nanoenzyme surface, the material exhibits a distinct core-shell structure, forming spherical nanoparticles (NPs).

[0045] Example 2

[0046] RAW264.7 and aHSCs cells were cultured in complete culture medium. Cell membrane proteins were extracted from these cells according to the kit instructions and then bound to nanomaterials. The nanomaterials were verified by SDS-PAGE electrophoresis according to the group order of RAW264.7, aHSCs, and HMCCNs.

[0047] Please see Figure 4 , Figure 4 This indicates that cell membrane characteristic proteins are expressed in HMCCNs, suggesting that the cell membrane has encapsulated nanomaterials.

[0048] Example 3

[0049] The same concentration of HCCNs material was irradiated with lasers of different power, and its temperature changes were recorded.

[0050] Please see Figure 5 , Figure 5 This indicates that the temperature of HCCNs increases with increasing laser power under different power irradiation, but the temperature remains at a low level.

[0051] Example 4

[0052] CNs and CCNs materials were added to H2O2 respectively, and compared with H2O2 alone, CNs, and CCNs materials to verify the catalytic strength of CCNs materials in H2O2 decomposition. Then, a kit was used to verify the CAT enzyme activity of CCNs materials.

[0053] Please see Figure 6 ,from Figure 6 It can be seen that CCNs can catalyze the production of a large number of bubbles from H2O2, while only a few bubbles appeared in the CNs and H2O2 groups, which confirms the effective catalytic activity of CCNs for H2O2 and that CCNs have CAT enzyme activity.

[0054] Example 5

[0055] After aHSC cells were plated, they were treated with TGF-β and incubated for 12 h. The original culture medium was removed, and the materials and cells were co-incubated again. After a certain incubation period, the original culture medium was discarded, and the cells were washed three times with PBS. ADHP probes were added and incubated for a period, followed by three washes with PBS. The cells were then fixed with 4% paraformaldehyde and gently washed three times with PBS to remove the fixative. Nuclei were stained with DAPI. Intracellular ADHP fluorescence was observed using a confocal laser scanning microscope (CLSM). The intracellular CAT enzyme activity of the CCNs materials was investigated.

[0056] Please see Figure 7 ,Depend on Figure 7 It is evident that as the concentration of CCNs increases, the fluorescence signal in aHSCs cells gradually weakens, indicating a decrease in intracellular reactive oxygen species and an increase in the activity of the CAT enzyme in the material.

[0057] Example 6

[0058] The RhB-labeled hybrid biomimetic nanodelivery system CCNs prepared in Example 1 of this invention, at a dose of 3 mg / kg, was intravenously injected into a mouse model of liver fibrosis. Analysis was performed using an in vivo imaging system 6 h after tail vein injection.

[0059] Please see Figure 8 ,Depend on Figure 8 It is evident that the carbon dot-doped nitride carbon hybrid biomimetic nanodelivery system prepared in this invention has a targeting effect on liver fibrosis, thereby achieving specific treatment for it.

[0060] The materials and instruments used in all the above embodiments are sourced from the materials listed in Table 1.

[0061] Table 1 shows the sources of the experimental materials and instruments used in the implementation of this application.

[0062] Experimental materials source Item number C57BL / 6 mice Nantong University Experimental Animal Center order melamine Aladdin M108433 carbon quantum dots Xianfeng Nano 102632 H2O2 Aladdin H433859 TGF-β Corning 354039 Cell membrane protein and cytoplasmic protein extraction kit Azure Sky P0033 Agarose Aladdin A118882 SDS-PAGE electrophoresis solution Azure Sky P0014A Loading buffer (5X) Azure Sky P0015 Coomassie Brilliant Blue G250 Azure Sky ST1119 DAPI Sigma Aldrich D9542 Rhb (Rhodamine B) Sigma Aldrich 83689 Small animal optical live imaging system PerkinElmer D8 Advance X-ray diffractometer Bruker AXS GmbH, Germany High-resolution transmission electron microscopy Thermo Fisher 808nm laser PURE Materials Laser confocal microscope Leica

[0063] The above description is merely an embodiment of this application and does not limit the patent scope of this application. Any equivalent structural or procedural transformations made using the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of this application.

Claims

1. A hybrid biomimetic nanodelivery system, characterized in that, The hybrid biomimetic nanodelivery system consists of a carbon dot-doped nitrogen-based carbon nanoparticle core and a cell membrane assembly that encapsulates the nitrogen-based carbon nanoparticle core.

2. The hybrid biomimetic nanodelivery system according to claim 1, characterized in that, The cell membrane group is composed of RAW264.7 cell membrane and aHSCs cell membrane in a ratio of 1:0.5 to 1, which can avoid immune clearance by the body.

3. The hybrid biomimetic nanodelivery system according to claim 1, characterized in that, The diameter of the nitrogen-based carbon nanoparticles is approximately 100-200 nm.

4. The hybrid biomimetic nanodelivery system according to claim 1, characterized in that, The hybrid biomimetic nanodelivery system has CAT enzyme activity to improve the hypoxic conditions of liver fibrotic tissue; the hybrid biomimetic nanodelivery system can achieve photothermal therapy for liver fibrosis at low temperatures.

5. A method for preparing a hybrid biomimetic nanodelivery system, characterized in that, include S1. The carbon dot precursor and the nitrogen-containing precursor are mixed and dried in an oven, and then calcined at high temperature under nitrogen protection to obtain carbon dot-doped carbon nitride (CCNs). S2. Disperse the carbon doped carbon nitride (CCN) powder in water and perform ultrasonic treatment to obtain a mixed solution; S3. The CCNs nanomaterials in the mixed solution are combined with the extracted RAW264.7 and aHSCs cell membrane proteins to obtain an HMCCNs hybrid biomimetic nanodelivery system.

6. The preparation method according to claim 5, characterized in that, In step S1, The nitrogen-containing precursor is melamine, and the carbon dot precursor is carbon quantum dots; the ratio of melamine to carbon quantum dots is 1:(0.05-0.1); the calcination temperature is 400-700 ℃; the calcination time is 4-8 h; and the heating rate is 3-10 ℃ / min.

7. The preparation method according to claim 5, characterized in that, In step S2, the ultrasonic treatment time is 2-5 hours and the ultrasonic power is 300W.

8. The preparation method according to claim 5, characterized in that, In step S3, the ratio of RAW264.7 to aHSCs cell membranes is 1:(0.5-1), the ratio of cell membrane complex to CCNs is 1:(0.5-10), and the sonication time is 2-5 h.

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