LDH nanocomposites loaded with cd105 nanobodies, methods of making and uses thereof

Abstract

The application belongs to the field of biological composite materials, and specifically discloses a CD105 nanobody loaded LDH nanocomposite material and a preparation method and application thereof. The nanocomposite material is prepared by loading CD105 nanobody on the surface of LDH containing ICG, and the preparation method comprises the following steps: ICG-LDH is prepared by a coprecipitation method, and then the surface of the ICG-LDH is functionalized with CD105Nb in an electrostatic adsorption mode, so as to construct a CD105Nb-ICG-LDH nanocomposite material. The prepared CD105Nb-ICG-LDH nanocomposite biomaterial has good photothermal effect and photodynamic effect, can realize active targeting by means of the surface CD105Nb, and can induct tumor cell apoptosis in vitro and inhibit tumor growth in vivo by means of the photothermal effect and photodynamic effect generated under ICG illumination, so as to further provide experimental basis and theoretical guidance for subsequent tumor treatment clinical experiments.

Description

Technical Field

[0001] This invention belongs to the field of biocomposite materials, specifically relating to LDH nanocomposite materials loaded with CD105 nanoantibodies, their preparation methods, and applications. Background Technology

[0002] With changes in living environment and increasing work pressure, the incidence of malignant tumors is showing a year-on-year upward trend, becoming the second leading cause of death worldwide after cardiovascular and cerebrovascular diseases. Traditional treatments for tumors mainly include surgical resection and radiotherapy / chemotherapy, but due to their limitations, they often not only fail to cure the tumor but also irritate it, causing recurrence and progression. Photodynamic therapy (PDT), as an emerging tumor treatment method, has advantages such as minimal invasiveness, mild adverse reactions, repeatable treatment, and low likelihood of developing drug resistance. PDT involves a photosensitizer (PS) that, under irradiation with light of a specific wavelength, transitions from its ground state to an excited state and undergoes energy transfer with oxygen molecules to generate singlet oxygen. 1 Multiple reactive oxygen species (ROS), primarily O2, exert cytotoxic effects by selectively killing tumor cells, thereby achieving the goal of tumor treatment. Photosensitizers, as a key element of phototherapy (PDT), significantly influence the efficacy of PDT and its clinical application and promotion. However, second-generation photosensitizers generally suffer from insufficient targeting, poor stability, and short half-lives, limiting the clinical application of PDT in tumor treatment. Therefore, research and development of photosensitizers is crucial. 1 Third-generation "functional photosensitizers" with high O2 quantum yield, good targeting, and high stability are an important direction for building PDT (Prophylactic Device Therapy) platforms in the future.

[0003] Nanobodies (Nb) are miniature antibodies with a molecular weight of only about 15 kDa, derived from single-domain variable region fragments of heavy chain antibodies from camelids and cartilaginous fish. Nanobodies overcome the limitations of conventional monoclonal antibodies (mAbs) in terms of their physicochemical properties for clinical applications while maintaining excellent antigen recognition and binding capabilities. Firstly, Nb exhibits higher stability under extreme conditions than traditional antibodies: studies have shown that after incubation at 37°C for one week, Nb retains over 80% of its binding activity to specific targets. Secondly, the variable region of heavy chain (VHH) domain of Nb is highly homologous to the human VH domain, resulting in lower immunogenicity and greater advantages for clinical applications. Furthermore, compared to traditional monoclonal antibodies, Nb offers a wider range of epitope binding, lower production costs, and ease of artificial modification. These unique advantages of Nb provide new insights for active targeted photodynamic therapy of tumors. CD105, also known as endoglin (ENG), is a co-receptor for several members of the TFG-β cytokine superfamily. It is highly expressed in proliferating vascular endothelial cells and lymphatic endothelial cells, but almost not expressed in normal tissues. During active angiogenesis, CD105 expression in endothelial cells is enhanced, especially in the angiogenesis zone of terminal vessels, where CD105 is highly expressed. The absence or functional deficiency of CD105 leads to impaired angiogenesis and revascularization. Given its crucial role in angiogenesis and homeostasis, CD105 has become a potential therapeutic target for pro-angiogenic and anti-angiogenic therapies in malignant tumors, diabetic complications, hemangiomas, and ischemic diseases. To date, research on the application of nitrogen (Nb) in prophylactic tumor treatment (PDT) is extremely limited, and research on using Nb to construct novel "functional photosensitizers" for active targeting to improve the efficacy of PDT tumor treatment remains lacking. Therefore, further investigation into the efficacy and related mechanisms of Nb-based nanoparticles for tumor-targeted photodynamic therapy will lay the foundation for third-generation "functional photosensitizers" and has important clinical significance for tumor treatment.

[0004] Indocyanine green (ICG) is a contrast agent that has been used clinically for many years, and recent studies have found that it also has the potential to become a photosensitizer. Research shows that under the induction of an 810 nm laser, ICG has a strong photodynamic killing effect on various tumor cells. Furthermore, it is relatively inexpensive, has few toxic side effects, and strong tissue penetration, making it a valuable material for development and utilization. Therefore, ICG is a relatively ideal photosensitizing material for tumor phototherapy (PDT). However, ICG still has shortcomings such as poor water solubility, short in vivo half-life, and lack of active targeting.

[0005] LDH is a type of material composed of positively charged magnesia-like layers, in which interlayer anions and water molecules can form a stable two-dimensional layered structure. Its general formula can be represented as [M...]. 1-x 2+ M x 3+ (OH)2 x+ (A m- ) x / m ·nH2O, where M 2+ Representing divalent metal cations such as Mg 2+ Zn 2+ Ni 2+ Ca 2+ Cu 2+ Wait, M 3+ Represents trivalent metal cations, such as Al 3+ Fe 3+ Co 3+ and Ga 3+ Given its unique structural characteristics, LDH can be used to load drug-active molecules onto the interlayer by replacing interlayer anions with different cations through ion exchange reactions. Simultaneously, targeted biomolecules can be loaded onto the surface of the layers via electrostatic interactions. Besides its advantages such as good biocompatibility, tunable structure and composition, ease of modification, and large drug loading capacity, LDH is also easily degraded and removed, low in cost, and relatively simple to synthesize, making it one of the most promising drug carriers for biomedical materials research.

[0006] Currently, there are no reports on the preparation of photosensitizers based on the combination of the aforementioned nanobodies, indocyanine green, and LDH. Summary of the Invention

[0007] This invention discloses an LDH nanocomposite material loaded with CD105 nanobodies, its preparation method, and its application. Based on the screened CD105 Nb nanobodies, the surface of ICG-LDH material is functionalized to construct a CD105 Nb-ICG-LDH nanocomposite photodynamic material with active targeting function. This new material can achieve active targeting by means of CD105 Nb on the surface, providing a new direction for the development of third-generation "functional photosensitizers".

[0008] In one aspect, the present invention provides an LDH nanocomposite material loaded with CD105 nanoantibody, wherein the nanocomposite material is prepared by loading CD105 nanoantibody onto the surface of LDH containing ICG.

[0009] Among them, LDH refers to layered bimetallic hydroxide, ICG refers to indocyanine green, and CD105 nanobody refers to CD105 nanobody Nb184 disclosed in CN106928355A or CD105 nanobody Nb168 disclosed in CN106928358A.

[0010] Preferably, the LDH is a layered zinc-aluminum hydroxide.

[0011] A second aspect of this invention provides a method for preparing the above-mentioned LDH nanocomposite material loaded with CD105 nanoantibodies, comprising the following steps:

[0012] (1) Thoroughly mix the aqueous solution of soluble divalent metal salt, the aqueous solution of soluble trivalent metal salt, and the aqueous solution of ICG to obtain a mixed solution;

[0013] (2) Add the mixed solution to the alkaline solution and mix to obtain a suspension;

[0014] (3) Centrifuge the suspension to separate the supernatant and precipitate. Resuspend the precipitate with water, wash the precipitate, and then resuspend and separate the precipitate with water 1-3 times. Then add water to obtain an LDH nanoparticle suspension containing ICG.

[0015] (4) CD105 recombinant plasmid was transformed into Escherichia coli BL21 to obtain CD105 Nb. CD105 Nb was purified and then an aqueous solution of CD105 Nb was prepared.

[0016] (5) The LDH nanoparticle suspension containing ICG is added to the CD105 Nb aqueous solution, mixed thoroughly, and allowed to stand to obtain an LDH nanocomposite material loaded with CD105 nanoantibody.

[0017] Preferably, the cation in the aqueous solution of the soluble divalent metal salt is Mg. 2+ Zn 2+ 、 Fe 2+ or Ca 2+ The anion is Cl. - NO 3- or SO4 2- The cation in the aqueous solution of the soluble trivalent metal salt is Al. 3+ Fe 3+ or Co 3+ The anion is Cl. - NO 3- or SO4 2- .

[0018] Preferably, in step (1), the concentration of the soluble divalent metal salt aqueous solution is 0.4-2.4 mol / L, the concentration of the soluble trivalent metal salt aqueous solution is 0.2-1.2 mol / L, the concentration of the ICG aqueous solution is 2-6 mmol / L, and the volume ratio of the soluble divalent metal salt aqueous solution, the soluble trivalent metal salt aqueous solution and the ICG aqueous solution is (2-10):(2-10):(1-10).

[0019] Preferably, in step (2), the OH in the alkaline solution... - The concentration is 0.05-0.45 mol / L, and the volume ratio of the alkaline solution to the mixed solution is (0.6-3):(3-6).

[0020] Preferably, step (4) specifically includes: plasmid transformation of *E. coli* BL21 DE3 competent cells using heat shock, followed by IPTG induction; collection of bacterial cells after induction, cell lysis, centrifugation of the lysate, purification of the supernatant of the lysed bacteria using a BeyoGold™ His-tag purification kit to obtain CD105 Nb nanobody stock solution, and adjustment of the CD105 Nb nanobody stock solution to a suitable concentration of CD105 Nb aqueous solution by adding dd H2O. More preferably, the optimal induction conditions for IPTG induction are: IPTG concentration of 0.1 mM, temperature of 15°C, and induction time of 6 h.

[0021] Preferably, in step (5), the LDH nanoparticle suspension containing ICG is added to the CD105 Nb aqueous solution, and the mass ratio of the LDH nanoparticles containing ICG to CD105 Nb is (5-200):1.

[0022] Preferably, in step (1), the soluble divalent metal salt is zinc chloride, and the soluble trivalent metal salt is aluminum chloride;

[0023] In step (2), the mixed solution is added to the alkaline solution under stirring conditions, and the mixing includes stirring for 0.5-5 hours.

[0024] In step (5), thorough mixing includes shaking and stirring for 0.4-12 hours.

[0025] A third aspect of the present invention provides a pharmaceutical composition comprising an LDH nanocomposite material loaded with CD105 nanobodies as described above, or an LDH nanocomposite material loaded with CD105 nanobodies prepared by the preparation method described above, and a pharmaceutically acceptable carrier.

[0026] The fourth aspect of this invention provides the application of the LDH nanocomposite material loaded with CD105 nanoantibodies as described above, or the LDH nanocomposite material loaded with CD105 nanoantibodies prepared by the preparation method described above, in the preparation of antitumor drugs.

[0027] The LDH nanocomposite material loaded with CD105 nanoantibody described above improves the stability of ICG and enhances its performance by relying on the LDH carrier. 1 The O2 generation rate relies on CD105 nanobody to improve the targeting of nanocomposite materials, thereby more accurately and efficiently targeting and killing actively proliferating tumor cells, reducing the number of administrations, and improving the efficacy of PDT for tumors.

[0028] The LDH nanocomposite material loaded with CD105 nanobodies described above can specifically and actively target tumor regions, and has the advantages of high efficiency, safety, stability and ideal half-life. The successful development of this nanocomposite material provides a novel treatment strategy for the field of tumor treatment. This strategy not only enhances the targeting and stability of drugs, but may also improve the treatment effect by regulating the tumor microenvironment, showing great potential for future clinical applications.

[0029] The preparation method of LDH nanocomposite material loaded with CD105 nanobodies described above was tested by ELISA. The loading rate of CD105 by ICG-LDH was detected, and the results showed that the loading efficiency of ICG could approach 100%.

[0030] Based on the analysis of UV-Vis spectroscopy, thermogravimetric analysis and thermal imaging, the LDH nanocomposite material loaded with CD105 nanobody has a higher singlet oxygen yield and better photothermal conversion efficiency compared with ICG.

[0031] The LDH nanocomposite material loaded with CD105 nanobodies described above has been confirmed by in vitro antitumor experiments to achieve active targeting through the CD105 Nb surface loading. It can also induce tumor cell apoptosis in vitro by generating photothermal and photodynamic effects under ICG irradiation, while inhibiting tumor growth in vivo. This provides experimental evidence and theoretical guidance for subsequent clinical trials of tumor treatment. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the preparation method of LDH nanocomposite material loaded with CD105 nanoantibody.

[0033] Figure 2 This is an SDS-PAGE analysis of CD105 Nb expression detection.

[0034] Figure 3A is the standard absorbance curve of CD105 concentration. Figure 3 B is a schematic diagram illustrating the load rate of ICG-LDH on CD105 detected by ELISA.

[0035] Figure 4 A is the TEM and AFM characterization diagram of LDH. Figure 4 B is the TEM and AFM characterization diagram of ICG-LDH(LI). Figure 4 C is the TEM and AFM characterization of CD105 Nb-ICG-LDH (LIN). A, B, and C, from left to right, are the TEM characterization (100 nm scale bar), the TEM characterization (50 nm scale bar), and the AFM characterization (200 nm scale bar).

[0036] Figure 5 A, 5B, and 5C are schematic diagrams showing the particle size, potential, and dispersity coefficient of CD105 Nb, LDH, ICG-LDH (LI), and CD105 Nb-ICG-LDH (LIN), respectively.

[0037] Figure 6 A is the Fourier transform infrared spectrum (FT-IR) of LDH, ICG-LDH (LI), and CD105 Nb-ICG-LDH (LIN). Figure 6 B is the X-ray diffraction (XRD) pattern of LDH, ICG-LDH (LI), and CD105 Nb-ICG-LDH (LIN). Figure 6 C is Figure 6 A magnified schematic diagram of the characteristic peak 006 in B.

[0038] Figures 7A, 7B, and 7C show the singlet oxygen yields of LDH, ICG, ICG-LDH (LI), and CD105 Nb-ICG-LDH (LIN) UV-Vis-NIR detection materials at optical power densities of 0.5 W / cm², 1 W / cm², and 2 W / cm², respectively. Figure 7D From left to right, the graphs show the singlet oxygen yield changes of LDH, ICG, ICG-LDH (LI), and CD105Nb-ICG-LDH (LIN) at optical power densities of 0.5 W / cm², 1 W / cm², and 2 W / cm².

[0039] Figure 8 A is the absorbance curve of ICG at different time points. Figure 8 B is the absorbance curve of CD105 Nb-ICG-LDH(LIN) at different time points. Figure 8 C is a statistical analysis graph of absorbance data of LIN and ICG at different time points.

[0040] Figure 9 A is a schematic diagram showing the temperature changes of CD105 Nb-ICG-LDH(LIN) (2.5µg / mL, 20µg / mL) and ICG (2.5µg / mL, 20µg / mL) under 730 nm laser light (optical power density 1 W / cm²). Figure 9 B is a thermal imaging thermogram of CD105 Nb-ICG-LDH(LIN) (2.5µg / mL, 20µg / mL) and ICG (2.5µg / mL, 20µg / mL).

[0041] Figure 10 The expression levels of CD105 on the surface of HepG2, MCF-7, and PANC-1 cells were detected by flow cytometry.

[0042] Figure 11 A is a schematic diagram illustrating the binding rates of ICG-LDH (LI), FAP Nb-ICG-LDH (Irr Nb-LIN), CD105Nb-ICG-LDH (CD105 Nb-LIN), CD105 Nb-ICG-LDH+ rCD105 (CD105 Nb-LIN+ CD105 ProBlock), and CD105 Nb-ICG-LDH+ light irradiation (CD105 Nb-LIN+ NIR) with target cells MCF-7, HepG2, and PANC-1, respectively, as determined by flow cytometry. Figure 11 B is a statistical analysis chart showing the binding rates of LI, Irr Nb-LIN, CD105 Nb-LIN, CD105 Nb-LIN+ CD105 ProBlock, and CD105 Nb-LIN+NIR to target cells MCF-7, HepG2, and PANC-1.

[0043] Figure 12 A shows the DAPI, Cy3, and Merge fluorescence staining images of HepG2 and PANC-1 cells in the LSCM-treated groups (LI, LIN, CD105 Nb-ICG-LDH, CD105 Nb-ICG-LDH + 1 W / cm² near-infrared light irradiation (LIN+L) and the blank control group (Control) for ICG-LDH (LI), CD105 Nb-ICG-LDH (LIN+L), and near-infrared light irradiation (LIN+L). Figure 12 B is a statistical analysis graph showing the binding rates of LI, LIN, LIN+L treatment groups and the Control group to HepG2 and PANC-1 cells as detected by LSCM. Figure 12 C is a schematic diagram illustrating the binding of LI, LIN, LIN+L treatment groups, and Control group to HepG2 and PANC-1 cells as detected by flow cytometry. Figure 12The graph shows the statistical analysis of the binding rates of HepG2 and PANC-1 cells by flow cytometry in the LI, LIN, LIN+L treatment groups and the Control group.

[0044] Figure 13 A shows the Hoechst, DCF, and Merge fluorescence staining images of HepG2 and PANC-1 cells in the LSCM-treated groups (LI), FAP Nb-ICG-LDH (Irr Nb-LIN), CD105 Nb-ICG-LDH (CD105 Nb-LIN), and CD105 Nb-ICG-LDH+ rCD105 (CD105 Nb-LIN+ CD105 Pro Block) and the blank control group. Figure 13 B is a single-line oxygen yield statistical analysis chart of the LI, Irr Nb-LIN, CD105 Nb-LIN, CD105 Nb-LIN + CD105 Pro Block (CD105 Pro Block) treatment groups and the Control group detected by LSCM. Figure 13 C is a schematic diagram illustrating the levels of intracellular ROS in HepG2 and PANC-1 cells detected by flow cytometry in the ICG-LDH (LI), FAPNb-ICG-LDH (Irr Nb-LIN), CD105 Nb-ICG-LDH (CD105 Nb-LIN), CD105 Nb-ICG-LDH+rCD105 (CD105 Nb-LIN+ CD105 Pro Block) treatment groups and the blank control group. Figure 13 D is a single-line oxygen yield statistical analysis graph of the LI, Irr Nb-LIN, CD105 Nb-LIN, CD105 Nb-LIN + CD105 Pro Block (CD105 Pro Block) treatment groups and the Control group detected by flow cytometry.

[0045] Figure 14 This is a schematic diagram illustrating the toxicity of ICG, ICG-LDH (LI), and CD105 Nb-ICG-LDH (LIN) to HUVECs cells under dark conditions using the CCK8 assay.

[0046] Figure 15 Figure A shows the toxicity of ICG, ICG-LDH (LI), and CD105Nb-ICG-LDH (LIN) to HUVECs cells after light exposure, as determined by the CCK8 assay. Figure 15Figure B shows the observation of the toxicity of ICG, ICG-LDH (LI), CD105Nb-ICG-LDH (LIN), and CD105-ICG-LDH) to HUVECs cells under light irradiation using Calcein-AM / PI staining.

[0047] Figure 16 Figure A shows flow cytometry results of apoptosis induced in MCF-7, HepG2, and PANC-1 target cells by PBS, ICG-LDH (LI), FAP Nb-ICG-LDH (Irr Nb-LIN), CD105 Nb-ICG-LDH (CD105 Nb-LIN), and CD105 Nb-ICG-LDH+ rCD105 (CD105 Pro-Block), respectively. Figure 16 B is a comparison of the apoptosis rates of MCF-7, HepG2, and PANC-1 target cells induced by different treatment groups.

[0048] Figure 17 These are images of the damage to target cells HepG2 after treatment with PBS, ICG-LDH (LI), FAP Nb-ICG-LDH (Irr Nb-LIN), CD105 Nb-ICG-LDH (CD105 Nb-LIN), and CD105 Nb-ICG-LDH+ rCD105 (CD105 Pro-Block) using transmission electron microscopy (magnification from top to bottom).

[0049] Figure 18 This is a schematic diagram of an in vitro treatment model for tumor-bearing mice.

[0050] Figure 19 The graphs show the effects of PBS, ICG-LDH (LI), FAP Nb-ICG-LDH (Irr Nb-LIN), CD105 Nb-ICG-LDH (CD105 Nb-LIN), and CD105 Nb-ICG-LDH+ rCD105 (CD105 Block) on the inhibition of subcutaneous transplanted tumor HepG2 growth. In the graphs, A is the tumor growth curve analysis, B is the weight analysis of tumor-bearing mice in each group, and C is the comparison of tumor quality of tumor-bearing mice in each group after sacrifice. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0052] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0053] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0054] This embodiment provides an LDH nanocomposite material loaded with CD105 nanoantibody, which is prepared by loading CD105 nanoantibody onto the surface of LDH containing ICG.

[0055] Among them, LDH refers to layered bimetallic hydroxide, ICG refers to indocyanine green, and CD105 nanobody refers to CD105 nanobody Nb184 disclosed in CN106928355A or CD105 nanobody Nb168 disclosed in CN106928358A.

[0056] Preferably, the LDH is a layered zinc-aluminum hydroxide.

[0057] This embodiment also provides a method for preparing the above-mentioned LDH nanocomposite material loaded with CD105 nanoantibody, combined with Figure 1 As shown, it includes the following steps:

[0058] (1) Thoroughly mix the aqueous solution of soluble divalent metal salt, the aqueous solution of soluble trivalent metal salt, and the aqueous solution of ICG to obtain a mixed solution;

[0059] (2) Add the mixed solution to the alkaline solution and mix to obtain a suspension;

[0060] (3) Centrifuge the suspension to separate the supernatant and precipitate. Resuspend the precipitate with water, wash the precipitate, and then resuspend and separate the precipitate with water 1-3 times. Then add water to obtain an LDH nanoparticle suspension containing ICG.

[0061] (4) CD105 recombinant plasmid was transformed into Escherichia coli BL21 to obtain CD105 Nb. CD105 Nb was purified and then an aqueous solution of CD105 Nb was prepared.

[0062] (5) The LDH nanoparticle suspension containing ICG is added to the CD105 Nb aqueous solution, mixed thoroughly, and allowed to stand to obtain an LDH nanocomposite material loaded with CD105 nanoantibody.

[0063] Preferably, the cation in the aqueous solution of the soluble divalent metal salt is Mg. 2+ Zn 2+ Fe 2+ or Ca 2+ The anion is Cl. - NO 3- or SO4 2- The cation in the aqueous solution of the soluble trivalent metal salt is Al. 3+ Fe 3+ or Co 3+ The anion is Cl. - NO 3- or SO4 2- .

[0064] Preferably, in step (1), the concentration of the soluble divalent metal salt aqueous solution is 0.4-2.4 mol / L (e.g., 0.4, 1.2, 1.6, 2.0 or 2.4 mol / L), the concentration of the soluble trivalent metal salt aqueous solution is 0.2-1.2 mol / L (e.g., 0.2, 0.4, 0.6, 1.0 or 1.2 mol / L), and the concentration of the ICG aqueous solution is 2-6 mmol / L (e.g., 2, 3.5, 4, 5 or 6 mmol / L, and the volume ratio of the soluble divalent metal salt aqueous solution, the soluble trivalent metal salt aqueous solution and the ICG aqueous solution is (2-10):(2-10):(1-10) (e.g., 2:2:1, 10:10:7, 5:5:5, 4:4:5 or 5:5:6).

[0065] Preferably, in step (2), the OH in the alkaline solution... - The concentration is 0.05-0.45 mol / L (e.g., 0.05, 0.10, 0.15, 0.25 or 0.45 mol / L), and the volume ratio of the alkaline solution to the mixed solution is (0.6-3):(3-6) (e.g., 0.6:3, 1.5:4, 0.6:4, 2:6 or 1.5:6).

[0066] Preferably, step (4) specifically includes: plasmid transformation of *E. coli* BL21 DE3 competent cells using heat shock, followed by IPTG induction; collection of bacterial cells after induction, cell lysis, centrifugation of the lysate, purification of the supernatant of the lysed bacteria using the BeyoGold™ Histtag purification kit to obtain CD105 Nb nanobody stock solution, and adjustment of the CD105 Nb nanobody stock solution to a suitable concentration of CD105 Nb aqueous solution by adding dd H2O. More preferably, the optimal induction conditions for IPTG induction are: IPTG concentration of 0.1 mM, temperature of 15°C, and induction time of 6 h.

[0067] Preferably, in step (5), the LDH nanoparticle suspension containing ICG is added to the CD105 Nb aqueous solution, and the mass ratio of the LDH nanoparticles containing ICG to CD105 Nb is (5-200):1 (e.g., 5:1, 20:1, 50:1, 100:1, 150:1 or 200:1).

[0068] Preferably, in step (1), the soluble divalent metal salt is zinc chloride, and the soluble trivalent metal salt is aluminum chloride;

[0069] In step (2), the mixed solution is added to the alkaline solution under stirring conditions, and the mixing includes stirring for 0.5-5 h (e.g., 0.5, 1, 2, 3 or 5 h).

[0070] In step (5), thorough mixing includes shaking and stirring for 0.4-12 hours (e.g., 0.4, 0.5, 2, 6, 10 or 12 hours).

[0071] The present invention will be further described below by way of examples, but the scope of protection of the present invention is not limited to these embodiments.

[0072] Example 1: Preparation of LDH nanocomposite material loaded with CD105 nanobody (CD105 Nb-ICG-LDH nanocomposite material)

[0073] The preparation method of CD105 Nb-ICG-LDH nanocomposite material includes the following steps:

[0074] (1) Take 5 mL of 1.2 mol / L ZnCl2 aqueous solution, 5 mL of 0.6 mol / L AlCl3 aqueous solution and 5 mL of 4 mmol / L ICG aqueous solution and put them into a small beaker, mix them thoroughly to obtain a mixed solution;

[0075] (2) Take 40 mL of 0.15 mol / L NaOH aqueous solution into an Erlenmeyer flask, place a rotor in it, and stir at room temperature and 1000 rpm. Transfer the mixed solution from the small beaker dropwise into the Erlenmeyer flask and continue to stir magnetically for 6 hours to obtain a suspension.

[0076] (3) Transfer the suspension to a centrifuge tube, centrifuge for 5 minutes, separate the supernatant and precipitate, resuspend the precipitate in 40 mL of ultrapure water, wash the precipitate, and centrifuge and wash twice more to remove Zn. 2+ Al 3+ After the salt ions are fully washed away, the precipitate is resuspended in 40 mL of ultrapure water to obtain an LDH nanoparticle suspension containing ICG (ICG-LDH suspension), which is then allowed to stand for later use.

[0077] (4) The CD105 recombinant plasmid was transformed into Escherichia coli BL21 to obtain CD105 Nb. The CD105 Nb was purified and an aqueous solution of CD105 Nb was prepared. Specifically, the CD105 recombinant plasmid was transformed into Escherichia coli BL21 (DE3). The BL21 (DE3) competent cells were transformed with plasmid by heat shock method and then induced with IPTG. After induction, the bacterial cells were collected and the cells were lysed. The lysate was centrifuged and the supernatant of the lysed bacteria was purified using the BeyoGold™ His-tag purification kit to obtain CD105 Nb nanobody stock solution. Then, dd H2O was added to adjust the CD105 Nb nanobody stock solution to an appropriate concentration of the CD105 Nb aqueous solution.

[0078] The IPTG induction conditions were as follows: IPTG concentration of 0.1 mM, temperature of 15℃, and induction time of 6 h. For purification, His-tag-labeled CD105 Nb was prepared and purified using nickel column affinity chromatography. The purification was verified by SDS-PAGE and Coomassie brilliant blue staining, and antibody purity was analyzed using ImageLab software. Western blot analysis was used to detect antibody expression. An aqueous solution of purified CD105 Nb was prepared for later use.

[0079] CD105 uses either the CD105 nanobody Nb184 disclosed in CN106928355A or the CD105 nanobody Nb168 disclosed in CN106928358A. This embodiment uses the CD105 nanobody Nb184.

[0080] (5) The LDH nanoparticle suspension containing ICG was added dropwise to the CD105 Nb aqueous solution of different initial concentrations, with the mass ratios of ICG-containing LDH nanoparticles to CD105 Nb being 5:1, 10:1, 20:1, 50:1, 100:1, and 200:1, respectively. The mixture was magnetically stirred at 1000 rpm for 0.5 h at room temperature and allowed to stand to obtain the LDH nanocomposite material loaded with CD105 nanoantibody. The prepared LDH nanocomposite material loaded with CD105 nanoantibody (CD105 Nb-ICG-LDH) was placed in a 50 mL centrifuge tube and stored at 4 ℃ for later use.

[0081] In the above preparation method, the order of preparing the CD105 Nb aqueous solution and the LDH nanoparticle suspension containing ICG can be interchanged or performed simultaneously.

[0082] Example 2 Characterization of LDH nanocomposite material loaded with CD105 nanobody (CD105 Nb-ICG-LDH nanocomposite material)

[0083] The following analysis will focus on the CD105 Nb-ICG-LDH nanocomposite material (LIN) using ICG, CD105 Nb, LDH, ICG-LDH (LI), and FAP Nb-ICG-LDH materials. ICG is the ICG used in step (1), CD105 Nb is the CD105 Nb obtained in step (4), and ICG-LDH (LI) is obtained by centrifuging and drying the precipitate from the ICG-LDH suspension in step (3). LDH is prepared by mixing 5 mL of 1.2 mol / L ZnCl2 aqueous solution and 5 mL of 0.6 mol / L AlCl3 aqueous solution, adding it to 40 mL of 0.15 mol / L NaOH aqueous solution under stirring at 1000 rpm at room temperature, continuing magnetic stirring for 6 hours, washing according to the method in step (3), and then drying. The preparation method of FAP Nb-ICG-LDH is the same as that of CD105 Nb-ICG-LDH nanocomposite material, but CD105 is replaced with FAP nanobody. For details of FAP nanobody, please refer to CN118903406A.

[0084] The CD105 Nb obtained after purification of CD105 in step (4) is as follows: Figure 2As shown, E1 and E2 are two samples after purification in step (4), CL is the sample obtained after transforming E. coli BL21 with CD105 recombinant plasmid, W1 is the sample obtained after heat shock treatment, W2 is the sample obtained after IPTG induction, and W3 is the sample obtained after cell lysis. SDS-PAGE detection of the protein purified by nickel agarose affinity chromatography showed that a clear band appeared at the corresponding position of the theoretical molecular weight of 20±2 kD, which preliminarily confirmed that CD105 was successfully purified and the purity was >90%.

[0085] The drug loading and adsorption rate of ICG-LDH for CD105 Nb were determined by the BCA method, and the loading rate of ICG-LDH for CD105 was determined by ELISA. Figure 3 A is the absorbance standard curve of CD 105, and the loading rate is as follows: Figure 3 As shown in Figure B, the LDH:CD105 Nb mass ratio ranges from 5:1 to 200:1. With the increase of the carrier ratio, the loading efficiency of ICG tends to 100%, with the highest efficiency reached at a ratio of 50:1, after which the change is not significant. The CD105 Nb-ICG-LDH nanocomposite material used in subsequent experiments was prepared with a mass ratio of 50:1 between LDH nanoparticles containing ICG and CD105 Nb.

[0086] The basic morphology and fundamental properties, including particle size, potential, and dispersity coefficient, of LDH, ICG-LDH(LI), and CD105 Nb-ICG-LDH(LIN) nanocomposites were characterized by transmission electron microscopy (TEM). The TEM and AFM characterizations of LDH, ICG-LDH(LI), and CD105 Nb-ICG-LDH(LIN) are as follows: Figure 4 A- Figure 4 As shown in Figure C, LDH (Layered Hydroxide) exhibits a typical hexagonal layered structure with clearly visible plate-like morphology, indicating good crystallinity. These plate-like structures are approximately 90 nm in size. Compared to LDH, ICG-LDH shows no significant difference. However, the CD105 Nb-ICG-LDH surface displays a more complex morphology, possibly composed of aggregates of multiple small particles. Obvious aggregation between particles is visible in the image. AFM results indicate a certain degree of surface roughness in CD105 Nb-ICG-LDH, attributed to the surface functionalization of CD105 Nb.

[0087] The particle size, zeta potential, and polydispersity index (PDI) of CD105 Nb, LDH, ICG-LDH (LI), and CD105 Nb-ICG-LDH (LIN) nanocomposites were analyzed using a Malvern particle size analyzer, and the characteristics are as follows: Figure 5 In A, CD105 Nb has a particle size of approximately 50 nm (particle aggregation); LDH has a particle size of approximately 70 nm; LI has a particle size of approximately 80 nm; and LIN has the largest particle size, approximately 120 nm. Figure 5 In component B, CD105 Nb has a potential of approximately -20 mV; LDH has a potential of approximately +20 mV; LI has a positive potential of approximately +20 mV; and LIN has a potential of approximately -10 mV, demonstrating that the surface of the CD105 Nb-ICG-LDH nanocomposite is fully loaded with CD105 Nb. Polydispersity index (PDI): ICG: approximately 0.5, indicating a wide particle size distribution; LDH: approximately 0.25, indicating a narrow particle size distribution; LI: approximately 0.4, indicating a medium particle size distribution; LIN: approximately 0.4, indicating a wide particle size distribution.

[0088] The crystal structure of the CD105 Nb-ICG-LDH nanocomposite was characterized using Fourier transform infrared spectroscopy and X-ray diffraction, combined with... Figure 6 As shown in Figure A, after the introduction of ICG, at 1430 cm... -1 The peak appearing at [location] corresponds to the -CH3 asymmetric stretching vibration of carbonate, a characteristic functional group of ICG. After surface grafting of CD105 Nb, the peak at 1000–1100 cm⁻¹... -1 The presence of CO stretching vibrations at this point confirms the successful synthesis of CD105 Nb-ICG-LDH. Figure 6 B and Figure 6 As shown in Figure C, the XRD results show that after ICG intercalation, the 006 characteristic peak shifts significantly to the left, indicating that the interlayer spacing of ICG-LDH is significantly increased. This suggests that after loading CD105 Nb onto the surface, the surface charge of CD105Nb-ICG-LDH changes, thereby affecting the stress distribution inside the crystal and consequently affecting the interlayer spacing.

[0089] The singlet oxygen yield and stability of CD105 Nb-ICG-LDH nanocomposites were detected using UV-Vis spectroscopy, and the photothermal properties of the CD105 Nb-ICG-LDH nanocomposites were analyzed using thermal imaging experiments. An ethanol solution of DPBF (2×10⁻⁶) was used. 5 mol / L) was added to solutions with different materials (1 mL, 1×10⁻⁶). 5mol / L), using different power densities (0.5, 1, 2 W / cm³). 2 The samples were irradiated with a 730 nm laser, and their absorption spectra were measured using a UV-Vis-NIR spectrophotometer (ICG concentration: 20 µg / mL). Figure 7 shows the absorbance changes of four different materials (LDH, ICG, LI, and LIN) at three different optical power densities (0.5 W / cm², 1 W / cm², and 2 W / cm²), as well as their absorbance changes at different irradiation times (0 to 10 minutes). The results show that as the power increases, the DPBF decrease rate at 410 nm increases significantly. Under the same irradiation power, the singlet oxygen yield of LI and LIN is significantly higher than that of ICG. Figure 8 The results of the absorbance stability test of ICG and LIN materials over time are presented. The solutions of ICG and LIN materials were stored in a refrigerator at 4°C for 1-4 days respectively. The absorbance of each material under 730 nm laser (optical power density 1 W / cm²) was measured at 0 days, 1 day, 2 days, 3 days and 4 days. LIN showed better photostability than ICG, and its absorbance changed less over time, indicating that LIN as a photothermal material may have a longer shelf life and more stable performance in practical applications. Figure 9 A is a schematic diagram showing the temperature changes of CD105 Nb-ICG-LDH(LIN) (2.5µg / mL, 20µg / mL) and ICG (2.5µg / mL, 20µg / mL) under 730 nm laser light (optical power density 1 W / cm²), indicating that at the same concentration, LIN experiences a faster temperature rise compared to ICG; the thermal imaging results are as follows. Figure 9 As shown in Figure B, the CD105 Nb-ICG-LDH (LIN) constructed in this embodiment has the same photothermal properties as ICG. Under the same concentration conditions, CD105 Nb-ICG-LDH has a better photothermal conversion efficiency.

[0090] Example 3: In vitro antitumor function of CD105 Nb-ICG-LDH nanocomposite material

[0091] 3.1 Combined experiment

[0092] In vitro experiments used CD105-negative cell line MCF-7, and CD105-positive cell lines HepG2 and PANC-1. Target cells were seeded in 24-well plates (1×10⁻⁶ cells / well). 5 Cells / well) were incubated overnight. First, LIN was added to the seeded cells. After 4 hours of incubation, cells were collected and washed three times with PBS for flow cytometry analysis. The expression level of CD105 on the target cell surface was as follows: Figure 10As shown: MCF-7 (0.7%), HepG2 (67.3%), PANC-1 (97.4%).

[0093] The groups are as follows: 1) LI group: 2μg ICG-LDH; 2) Irrelevant control antibody group: 2μg FAP Nb-ICG-LDH (IrrNb-LIN); 3) Experimental group: CD105 Nb-ICG-LDH: 2μg CD105 Nb-ICG-LDH (CD105 Nb-LIN); 4) Target cell CD105 blocking group: CD105 Nb-ICG-LDH + rCD105: 2μg CD105 Nb-ICG-LDH + rCD105 3μL (according to the instructions) (CD105 Nb-LIN + CD105 Pro Block); 5) Experimental group + light irradiation: CD105 Nb-ICG-LDH: 2μg CD105 Nb-ICG-LDH + 1 W / cm² near-infrared light irradiation for 5 min (CD105 Nb-LIN + NIR). Add 1 mL of PBS, vortex to mix, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and wash 3 times. Add 2 μL of anti-his tag antibody, and use flow cytometry to detect the binding of each group of materials to MCF-7, HepG2, and PANC-1, and analyze the binding rate. Analyze the data using FlowJo software. The results are as follows: Figure 11 A and Figure 11 As shown in Figure B, CD105 Nb-ICG-LDH can specifically bind to target cells HepG2 and PANC-1. This specific binding depends on the expression level of CD105 on the surface of the target cells. Furthermore, the binding can be specifically blocked by CD105 protein, demonstrating the specificity of the targeting function. In addition, this binding is relatively stable, and near-infrared light irradiation does not affect its binding efficiency.

[0094] 3.2 Targeted Delivery Experiment

[0095] Target cells HepG2 and PANC-1 were seeded into 12-well plates (2 × 10⁻⁶ cells per well). 5Cells / well were cultured overnight. Then, HepG2 and PANC-1 cells were added to the corresponding materials containing Cy3-dsDNA and CG-LDH culture medium. After 4 h of incubation, half of the cells were collected, washed three times with PBS, fixed with 4% paraformaldehyde, stained with DAPI, and captured by LSCM. The other half was analyzed by flow cytometry. The groups were as follows: 1) Blank control group: no material added (Control); 2) LI group: 2 μg ICG-LDH; 3) Experimental group: CD105 Nb-ICG-LDH: 2 μg CD105 Nb-ICG-LDH (LIN); 4) Experimental group + light: 2 μg CD105 Nb-ICG-LDH + 1 W / cm² near-infrared light irradiation for 5 min (LIN+L). Results are as follows. Figure 12 As shown, Figure 12 A shows the DAPI, Cy3, and Merge fluorescence staining images of HepG2 and PANC-1 cells in the near-infrared light irradiation (LIN+L) treatment group and the blank control group (Control) after LSCM detection of ICG-LDH (LI), CD105 Nb-ICG-LDH (LIN), CD105 Nb-ICG-LDH+1 W / cm², and LIN+L treatment. Figure 12 B is a statistical analysis graph showing the binding rates of LI, LIN, LIN+L treatment groups and the Control group to HepG2 and PANC-1 cells as detected by LSCM. Figure 12 C is a schematic diagram illustrating the binding of LI, LIN, LIN+L treatment groups, and Control group to HepG2 and PANC-1 cells as detected by flow cytometry. Figure 12 Statistical analysis of the binding rates of HepG2 and PANC-1 cells by flow cytometry in the LI, LIN, LIN+L treatment groups and the Control group; Figure 12 The results show that CD105 Nb-ICG-LDH, after being loaded onto the CD105 Nb surface, can be more efficiently targeted to cells than ICG-LDH, thus increasing the rate at which cells take up the material.

[0096] 3.3 Intracellular reactive oxygen species experiment

[0097] Target cells HepG2 and PANC-1 were seeded into 24-well plates (1 × 10⁻⁶ cells per well). 5Cells / well) and cultured overnight. Materials were added to each group as follows: 1) LI group: 2 μg ICG-LDH; 2) Irrelevant control antibody group: 2 μg FAP Nb-ICG-LDH (Irr Nb-LIN); 3) Experimental group: CD105 Nb-ICG-LDH: 2 μg CD105 Nb-ICG-LDH (CD105 Nb-LIN); 4) Target cell CD105 blocking group: CD105 Nb-ICG-LDH + rCD105: 2 μg CD105 Nb-ICG-LDH + rCD105 3 μL (according to the manufacturer's instructions) (CD105 Nb-LIN + CD105 Pro Block); 5) Blank control group: no materials added (Control). Cells were cultured for another 6 hours, then incubated with the singlet oxygen probe DCFH-DA (10 µM) for 20 minutes, followed by irradiation with near-infrared light at 1 W / cm² for 5 minutes and Hoechst staining. Finally, fluorescence images were captured in LSCM and flow cytometry data were analyzed. Results are as follows: Figure 13 As shown in AD, where... Figure 13 A shows that cells treated with CD105 Nb-LIN exhibited significant green fluorescence, indicating substantial ROS production; the fluorescence intensity of the CD105 Nb-LIN + CD105 Pro-Block group was significantly reduced, indicating that the blocking agent effectively inhibited the material's capture of target cells, thereby limiting ROS production; the control group showed almost no fluorescence, indicating a very low basal ROS level. Figure 13 As shown in C, the CD105 Nb-LIN treatment group showed high fluorescence intensity in both HepG2 and PANC-1 cell lines, indicating that the treatment significantly increased the intracellular ROS level. After the addition of CD105 protein blockade, the fluorescence intensity decreased, indicating the specificity of the target. Figure 13 Statistical analysis of the average fluorescence intensity of each group by D proved that the difference was statistically significant.

[0098] 3.4 In vitro toxicity tests (under dark and light conditions)

[0099] HUVECs (CD105-positive cells) were seeded in 96-well plates (5000 cells / well) and cultured overnight. Next, fresh DMEM medium containing ICG, ICG-LDH, and CD105 Nb-ICG-LDH (ICG = 0–20 µg / mL) was added to replace the previous medium, and the cells were cultured for another 24 hours. Dark environment group: Cells were collected and washed three times with PBS, then dispersed in fresh medium. The cells were then cultured for another 6 hours, and their viability was quantified using the CCK8 assay. Figure 14 Illuminated group: Cells were collected and washed three times with PBS, then dispersed in fresh culture medium. The cells were then cultured for another 6 hours and treated with an 808nm laser (0.5W / cm²). 2 Irradiate for 5 minutes ( Figure 15 Finally, cell viability was analyzed and quantified using the CCK8 assay. Cells in the light-treated group were simultaneously labeled using a Calcein-AM / PI double staining kit (AM:PI:buffer = 1:1:1000), incubated at 37°C in the dark for 35 min, and observed using LSCM. Figure 14 As shown, under dark conditions, CCK8 results indicated that ICG, ICG-LDH, and CD105Nb-ICG-LDH had no significant effect on cell viability. (Combined with...) Figure 15 A and Figure 15 As shown in Figure B, after near-infrared light irradiation, both CCK8 and LSCM results showed that cell viability decreased significantly with increasing concentrations of ICG-LDH and CD105 Nb-ICG-LDH, and the proportion of dead cells in the CD105 Nb-ICG-LDH treatment group increased more significantly (2.5–20 µg / mL).

[0100] 3.5 In vitro apoptosis experiment

[0101] Target cells MCF-7, HepG2, and PANC-1 were seeded in 24-well plates (1×10⁻⁶ cells / well). 5 Cells / well) and cultured overnight. Materials were added to each group as follows: 1) LI group: 2 μg ICG-LDH; 2) Irrelevant control antibody group: 2 μg FAP Nb-ICG-LDH (Irr Nb-LIN); 3) Experimental group: CD105 Nb-ICG-LDH: 2 μg CD105 Nb-ICG-LDH (CD105 Nb-LIN); 4) Target cell CD105 blocking group: CD105 Nb-ICG-LDH + rCD105: 2 μg CD105 Nb-ICG-LDH + rCD105 3 μL (according to the instructions) (CD105 Pro Block); 5) Blank control group: no materials added (PBS). 808nm laser (0.5W / cm²) 2 Irradiate for 5 minutes, collect cells, resuspend cells in 1× binding buffer, and adjust cell density to 1-5×10⁻⁶. 6 / ml. Take 100µl of cell suspension into a 5ml flow cytometry tube, add 5µl of rh Annexin V Alexa Fluor 647, gently mix with a pipette, and incubate at room temperature in the dark for 5 min. Finally, add 10µl of PI and 400µl of 1×PBS buffer, and immediately perform flow cytometry analysis. Flow cytometry results are shown below. Figure 16 As shown in A and 16B, CD105 Nb-ICG-LDH can significantly induce apoptosis in CD105-positive target cells. Blocking the antibody on the surface of CD105 through the protein can significantly reduce the death rate of CD105-positive target cells, indicating that this targeted killing effect is specific. Figure 17 As shown, combined with cell electron microscopy results, such as Figure 17 As shown, cells treated with CD105 Nb-ICG-LDH exhibited irregular morphology, cell membrane rupture, swollen or even absent mitochondria, and condensed chromatin in the cell nucleus.

[0102] 3.6 In vivo antitumor function of CD105 Nb-ICG-LDH nanocomposite material

[0103] 3.6.1 Test Methods

[0104] Combination Figure 18 As shown, HepG2 cells in good logarithmic growth phase were collected, washed 2-3 times with PBS, and then counted. The density of the cell suspension was adjusted to 1×10⁻⁶. 7 per mL.

[0105] Take 4-6 week old nude mice and subcutaneously inject 200 μL of HepG2 cells (generally 2 × 10⁻⁶ cells) near the right axilla. 6 (each individual), observe the tumor formation status, including tumor volume, tumor emergence rate, and tumor formation time.

[0106] The tumor size was measured regularly, and the subcutaneous tumor in the mice was kept at 50 mm. 3 Mice with consistent tumor characteristics and size were selected and randomly divided into 5 groups of 5 mice each to observe the therapeutic effect of the drug on mouse tumors. This period was recorded as (day 0). The experimental groups were as follows: 1) PBS group; 2) ICG-LDH group (LI); 3) FAP Nb-ICG-LDH (IrrNb-LI, irrelevant nanobody control); 4) CD105 Nb-ICG-LDH (LIN, experimental group); 5) CD105 Nb-ICG-LDH + rCD105 (CD105 Pro Block, blocking experimental group).

[0107] Interventions were administered according to the treatment methods for each group: Each experimental group received a tail vein injection of a drug containing 1.2 mg / kg ICG, while the PBS group received an equal volume of PBS solution. Six hours after injection, the tumor sites of mice were irradiated with near-infrared light (808 nm laser, power 0.5 W / cm²). 2Photodynamic therapy was performed, and the above procedure was repeated on days 2, 4, and 6 after injection. During and after treatment, tumor volume was measured regularly (e.g., every other day) to monitor tumor growth. Data from each measurement was recorded for subsequent analysis.

[0108] Treatment begins on day 0. Every 3 days, the length (L) and width (W) of the tumor are measured and recorded using calipers or a ruler, according to the formula V(mm). 3 )=W 2 ×L / 2. (V: tumor volume, W: width, L: length). Treatment ended when the maximum tumor length reached 15 mm. Blood was collected from the mice's eyes, the tumor was dissected, fat was removed, and the tumor weight was measured. The tumor weight inhibition rate was calculated as: tumor weight - average tumor weight of the treatment group / control group. A growth curve was plotted based on the tumor volume data. Survival time was recorded for another group of mice to analyze their survival rate. Daily food intake, body weight, and tumor diameter were recorded. Once the tumor size reached 2000 mm... 3 At that time, the mice were euthanized.

[0109] The results showed that there were no significant abnormalities in the mice's diet, activity, defecation, and respiration during the treatment and observation period, and there was no statistically significant difference in body weight among the groups. Figure 19 B). Before treatment, there was no statistically significant difference in tumor volume among the groups. During the observation period, tumor growth curves were plotted based on tumor volume, such as... Figure 19 In group A, the tumor volume of mice in each group continued to increase; however, treatment with CD105 Nb-ICG-LDH significantly slowed tumor growth. After the observation period, mice were sacrificed, and the tumors were weighed and analyzed. Figure 19 C showed that CD105 Nb-ICG-LDH had a significantly stronger anti-tumor effect than other treatment groups, and the difference was statistically significant.

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in each embodiment, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the technical solutions to deviate from the embodiments of the present invention.

Claims

1. A LDH nanocomposite loaded with CD105 nanobodies, characterized in that: the nanocomposite is prepared by loading CD105 nanobodies on the surface of LDH containing ICG; the preparation method of the nanocomposite comprises the following steps: (1) mixing a water-soluble divalent metal salt solution, a water-soluble trivalent metal salt solution and an ICG aqueous solution to obtain a mixed solution; (2) adding the mixed solution to an alkali solution to obtain a suspension; (3) centrifuging the suspension to separate the supernatant and the precipitate, resuspending the precipitate with water, washing the precipitate, resuspending and separating the precipitate for 1-3 times with water, and then adding water to obtain a LDH nanoparticle suspension containing ICG; (4) transforming E. coli BL21 with a CD105 recombinant plasmid to obtain CD105 Nb, and preparing a CD105 Nb aqueous solution after purifying the CD105 Nb; (5) adding the LDH nanoparticle suspension containing ICG to the CD105 Nb aqueous solution, mixing thoroughly, and standing to obtain the LDH nanocomposite loaded with CD105 nanobodies; in step (1), the water-soluble divalent metal salt is zinc chloride, and the water-soluble trivalent metal salt is aluminum chloride; in step (5), the LDH nanoparticle suspension containing ICG is added to the CD105 Nb aqueous solution, and the mass ratio of the LDH nanoparticle containing ICG to the CD105 Nb is (5-200):

1. 2.The LDH nanocomposite loaded with CD105 nanobodies according to claim 1, characterized in that: in step (1), the concentration of the water-soluble divalent metal salt solution is 0.4-2.4 mol / L, the concentration of the water-soluble trivalent metal salt solution is 0.2-1.2 mol / L, and the concentration of the ICG aqueous solution is 2-6 mmol / L, and the volume ratio of the water-soluble divalent metal salt solution, the water-soluble trivalent metal salt solution and the ICG aqueous solution is (2-10):(2-10):(1-10). 3.The LDH nanocomposite loaded with CD105 nanobodies according to claim 2, characterized in that: In step (2), the concentration of OH - in the alkali solution is 0.05-0.45 mol / L, and the volume ratio of the alkali solution to the mixed solution is (0.6-3):(3-6). 4.The LDH nanocomposite loaded with CD105 nanobodies according to claim 1, characterized in that: in step (2), the adding of the mixed solution to the alkali solution is carried out under stirring, and the mixing includes stirring for 0.5-5 h; in step (5), the thorough mixing includes oscillation stirring for 0.4-12 h. 5.A pharmaceutical composition comprising the LDH nanocomposite loaded with CD105 nanobodies according to any one of claims 1-4 and a pharmaceutically acceptable carrier. 6.The use of the LDH nanocomposite loaded with CD105 nanobodies according to any one of claims 1-4 in the preparation of an antitumor drug.

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