Nano complex as well as preparation method and application thereof

Abstract

The invention relates to the technical field of tumor treatment, in particular to a nano complex as well as a preparation method and application thereof. The nano complex provided by the invention comprises hollow mesoporous silicon dioxide, a fluorocarbon chain doped in a shell skeleton of the hollow mesoporous silicon dioxide, CaO2 loaded in a cavity of the hollow mesoporous silicon dioxide, and a CD105 targeting ligand modified on the surface of the hollow mesoporous silicon dioxide through a biological membrane bionic coating. Furthermore, a Toll-like receptor 7 / 8 dual-agonist can also be contained in the cavity of the hollow mesoporous silica. The nano complex provided by the invention not only can effectively relieve the hypoxia and acidic microenvironment of tumors, but also can enhance the anti-tumor immune response through immunoregulation and improve the tumor treatment effect. Moreover, the medicine can be accurately targeted to tumor tissues, accumulation of the medicine at tumor parts is improved, and side effects on normal tissues are reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor treatment, and in particular to a nanocomplex and a preparation method and application thereof. Background Art

[0002] The tumor microenvironment (TME) is a key factor in the survival and development of tumor cells. Its unique physiological and biochemical characteristics have an important impact on tumor growth, invasion, metastasis and treatment resistance. The tumor microenvironment is usually characterized by hypoxia and acidosis, which is caused by insufficient blood supply and metabolic disorders due to the rapid proliferation and abnormal vascularization of tumor cells. The hypoxic microenvironment not only promotes the invasion and metastasis of tumor cells, but also leads to increased resistance to radiotherapy and chemotherapy. In addition, the acidic microenvironment further weakens the body's anti-tumor immune response by inhibiting the function of immune cells. Therefore, how to effectively alleviate the hypoxic and acidic microenvironment of the tumor has become an important research direction in the current field of tumor treatment. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a nanocomplex and its preparation method and application. The nanocomplex provided by the present invention can effectively alleviate the hypoxia and acidic microenvironment of tumors.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a nanocomplex, comprising hollow mesoporous silica; the interior of the hollow mesoporous silica is a cavity, and the exterior is a shell with a mesoporous structure; the nanocomplex also includes a carbon fluorine chain doped in the hollow mesoporous silica shell skeleton, CaO2 loaded in the hollow mesoporous silica cavity, and a CD105 targeting ligand modified on the surface of the hollow mesoporous silica by a biomembrane biomimetic coating; the carbon fluorine chain is formed by hydrolysis of perfluoroalkylsiloxane.

[0006] Preferably, the cavity of the hollow mesoporous silica further comprises a Toll-like receptor 7 / 8 dual agonist.

[0007] Preferably, the Toll-like receptor 7 / 8 dual agonist comprises resiquimod.

[0008] Preferably, the biomembrane biomimetic coating comprises a lipid layer, a cell membrane or red blood cells.

[0009] Preferably, the CD105 targeting ligand comprises an anti-CD105 monoclonal antibody or a nucleic acid aptamer.

[0010] Preferably, the perfluoroalkylsiloxane includes 1H,1H,2H,2H-perfluorodecyltriethoxysilane.

[0011] Preferably, the pore diameter of the hollow mesoporous silica is 2 to 10 nm, the cavity diameter of the hollow mesoporous silica is 50 to 300 nm, and the shell thickness is 10 to 30 nm.

[0012] The present invention provides a method for preparing the nanocomplex described in the above scheme, comprising the following steps:

[0013] Mixing water, alcohol, ammonia water and a first silicate, performing a first hydrolysis and polycondensation to form a silica core, and obtaining a core dispersion; adding a mixture of perfluoroalkylsiloxane and a second silicate to the core layer dispersion, performing a second hydrolysis and polycondensation to form a silica shell containing a fluorine-carbon chain to coat the silica core, and collecting colloidal nanoparticles having a core-shell structure; removing the silica core from the colloidal nanoparticles having the core-shell structure to obtain FHMON; the FHMON comprises hollow mesoporous silica and carbon-fluorine chains doped in the hollow mesoporous silica shell skeleton;

[0014] A water-soluble calcium salt, the FHMON, water and a protective agent are mixed, and an aqueous hydrogen peroxide solution is added to the resulting mixture to carry out an oxidation reaction, thereby in situ forming CaO2 in the cavity of the hollow mesoporous silica. The pH value of the resulting oxidation product system is adjusted to 11-12, and after solid-liquid separation and washing, CaO2@FHMON nanoparticles are obtained;

[0015] Modifying the surface of the CaO2@FHMON nanoparticles with a biofilm-like coating to obtain CaO2@FHMON@Lip;

[0016] The CaO2@FHMON@Lip is dispersed in a polar organic solvent, and a NaHCO3 solution and a CD105 targeting ligand are sequentially added to the obtained dispersion. The CD105 targeting ligand is combined with the biomembrane biomimetic coating, and the nanocomplex is obtained after solid-liquid separation.

[0017] Preferably, when the cavity of the hollow mesoporous silica also includes a Toll-like receptor 7 / 8 dual agonist, the method further includes: dispersing CaO2@FHMON nanoparticles and the Toll-like receptor 7 / 8 dual agonist in a polar organic solvent, allowing the Toll-like receptor 7 / 8 dual agonist to enter the cavity of the hollow mesoporous silica, performing solid-liquid separation to obtain Toll / CaO2@FHMON, and then modifying the surface of the Toll / CaO2@FHMON with a biomembrane biomimetic coating.

[0018] The present invention provides the use of the nanocomplex described in the above scheme or the nanocomplex prepared by the preparation method described in the above scheme in the preparation of anti-tumor products.

[0019] The nanocomplex provided by the present invention comprises hollow mesoporous silica; the interior of the hollow mesoporous silica is a cavity, and the exterior is a shell with a mesoporous structure. The nanocomplex also includes a fluorocarbon chain doped within the hollow mesoporous silica shell skeleton, CaO2 loaded within the hollow mesoporous silica cavity, and a CD105 targeting ligand modified on the surface of the hollow mesoporous silica via a biofilm-inspired coating. The fluorocarbon chain is formed by the hydrolysis of perfluoroalkylsiloxane. The fluorocarbon chain can adsorb oxygen, carry the adsorbed oxygen to the tumor site, and release it to alleviate tumor hypoxia. The CaO2 can react with water to generate oxygen, further alleviating tumor hypoxia. The generated calcium hydroxide can neutralize the acidity of the tumor microenvironment, inhibiting tumor growth and metastasis. The CD105 targeting ligand can precisely guide the nanocomplex to tumor tissue, increasing drug accumulation at the tumor site and reducing side effects on normal tissues.

[0020] Furthermore, the nanocomplex provided by the present invention also includes a Toll-like receptor 7 / 8 dual agonist in the cavity of the hollow mesoporous silica, which can activate Toll-like receptors 7 / 8, promote the transformation of M2 macrophages into M1 macrophages, and enhance anti-tumor immune response. Therefore, the nanocomplex provided by the present invention can not only effectively alleviate the hypoxia and acidic microenvironment of the tumor, but also enhance the anti-tumor immune response through immunomodulation, thereby improving the tumor treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the preparation of the AL@O2-FMRC nanocomplex in Example 1 of the present invention;

[0022] Figure 2 UV-visible spectra of FM and FMRC;

[0023] Figure 3 Zeta potential and particle size distribution of FM, FMRC, L@FMRC, and AL@FMRC;

[0024] Figure 4 Schematic diagram of the structure of AL@FMRC and transmission electron microscopy and elemental mapping;

[0025] Figure 5 The graph shows the oxygen generation capacity test results of the blank control group, AL@O2-FMC, AL@FM, AL@O2-FMR and AL@O2-FMRC;

[0026] Figure 6 Schematic diagram of oxygen generation by AL@O2-FMRC;

[0027] Figure 7The following are morphological images of macrophages of different forms under a microscope;

[0028] Figure 8 The immunomodulatory ability test results of AL@O2-FMC and AL@O2-FMRC are shown in the figure;

[0029] Figure 9 The results of the tumor targeting ability test of AL@O2-FMRC and L@O2-FMRC are shown. DETAILED DESCRIPTION

[0030] The present invention provides a nanocomplex comprising hollow mesoporous silica, a carbon fluorine chain doped in a shell skeleton of the hollow mesoporous silica, CaO2 loaded in the cavity of the hollow mesoporous silica, and a CD105 targeting ligand modified on the surface of the hollow mesoporous silica by a biomimetic coating; the carbon fluorine chain is formed by hydrolysis of perfluoroalkylsiloxane.

[0031] The nanocomplex provided by the present invention includes hollow mesoporous silica. In the present invention, the interior of the hollow mesoporous silica is a cavity, and the exterior is a shell with a mesoporous structure. The pore diameter of the hollow mesoporous silica is preferably 2 to 10 nm, and in specific embodiments, it can be 2 nm, 4 nm, 6 nm, 8 nm, or 10 nm. The cavity diameter is preferably 50 to 300 nm, and in specific embodiments, it can be 50 nm, 100 nm, 150 nm, 200 nm, 250 nm, or 300 nm. The shell thickness is preferably 10 to 30 nm, and in specific embodiments, it can be 10 nm, 20 nm, or 30 nm. In the present invention, the hollow mesoporous silica serves as a carrier.

[0032] The nanocomplex provided by the present invention includes a fluorocarbon chain doped in the hollow mesoporous silica shell skeleton; the fluorocarbon chain is formed by hydrolysis of a perfluoroalkylsiloxane. In the present invention, the perfluoroalkylsiloxane preferably includes 1H,1H,2H,2H-perfluorodecyltriethoxysilane (PDES). In the present invention, the fluorocarbon chain has the function of adsorbing oxygen, and the adsorption amount is 0.5 to 1.5 mL of oxygen per mg of fluorocarbon chain. After the fluorocarbon chain carries oxygen to the tumor site, it can significantly increase the oxygen concentration in the tumor site, alleviate tumor hypoxia, and enhance the effect of radiotherapy and chemotherapy. The present invention has no special requirements for the doping amount of the fluorocarbon chain, and those skilled in the art can adjust it according to actual needs. For example, when it is necessary to carry more oxygen, the doping amount of the fluorocarbon chain can be appropriately increased, but the premise is to ensure the structural integrity of the hollow mesoporous silica.

[0033] The nanocomplex provided by the present invention includes CaO2 loaded in the hollow mesoporous silica cavity; in the present invention, the CaO2 can react with water to generate oxygen, further alleviating tumor hypoxia, and the calcium hydroxide generated at the same time can neutralize the acidity of the tumor microenvironment and inhibit tumor growth and metastasis. In the present invention, the oxygen generation amount of CaO2 is 0.2 to 0.5 mL of oxygen per mg of CaO2. The present invention does not impose any special restrictions on the loading amount of the CaO2, and those skilled in the art can make appropriate adjustments based on actual needs. In an embodiment of the present invention, the loading amount of the CaO2 is 0.95% (referring to the percentage of the mass of CaO2 to the mass of the nanocomplex).

[0034] The nanocomplex provided by the present invention comprises a CD105 targeting ligand modified on the surface of the hollow mesoporous silica through a biomembrane biomimetic coating.

[0035] In the present invention, the biomimetic layer preferably comprises a lipid layer, a cell membrane, or a red blood cell. The lipid layer is further preferably DPPC (dipalmitoylphosphatidylcholine) and DSPE-PEG2000-NHS (distearylphosphatidylethanolamine-polyethylene glycol 2000-N-hydroxysuccinimide ester); the mass ratio of DPPC to DSPE-PEG2000-NHS is preferably 4:7. The cell membrane preferably comprises a cancer cell membrane.

[0036] In the present invention, the CD105 targeting ligand refers to a ligand that targets the CD105 protein; the CD105 targeting ligand preferably comprises an anti-CD105 monoclonal antibody or a nucleic acid aptamer. In the embodiments of the present invention, the anti-CD105 monoclonal antibody is purchased from Abcam. In the present invention, the CD105 targeting ligand can precisely target tumor tissue, increase drug accumulation at the tumor site, and reduce side effects on normal tissues.

[0037] In the present invention, the cavity of the hollow mesoporous silica preferably also includes a Toll-like receptor 7 / 8 dual agonist; the Toll-like receptor 7 / 8 dual agonist preferably includes resiquimod (i.e., R848). In the present invention, the Toll-like receptor 7 / 8 dual agonist can activate Toll-like receptor 7 / 8, promote the transformation of M2 macrophages to M1 macrophages, and enhance anti-tumor immune response. The present invention does not specifically limit the drug loading of the Toll-like receptor 7 / 8 dual agonist, and those skilled in the art can appropriately adjust it according to actual needs. In an embodiment of the present invention, the drug loading of the Toll-like receptor 7 / 8 dual agonist is 2.13% (referring to the percentage of the mass of the Toll-like receptor 7 / 8 dual agonist to the mass of the nanocomplex).

[0038] The nanocomplex provided by the present invention has the function of actively targeting tumor cells and releasing oxygen in tumor cells under ultrasound, which can fully inhibit the production of lactic acid in the tumor microenvironment, enhance anti-tumor immunity, and achieve the purpose of radically curing tumors.

[0039] The present invention provides a method for preparing the nanocomplex described in the above scheme, comprising the following steps:

[0040] Mixing water, alcohol, ammonia water and a first silicate, performing a first hydrolysis and polycondensation to form a silica core, and obtaining a core dispersion; adding a mixture of perfluoroalkylsiloxane and a second silicate to the core layer dispersion, performing a second hydrolysis and polycondensation to form a silica shell containing a fluorine-carbon chain to coat the silica core, and collecting colloidal nanoparticles having a core-shell structure; removing the silica core from the colloidal nanoparticles having the core-shell structure to obtain FHMON; the FHMON comprises hollow mesoporous silica and carbon-fluorine chains doped in the hollow mesoporous silica shell skeleton;

[0041] A water-soluble calcium salt, the FHMON, water and a protective agent are mixed, and an aqueous hydrogen peroxide solution is added to the resulting mixture to carry out an oxidation reaction, thereby in situ forming CaO2 in the cavity of the hollow mesoporous silica. The pH value of the resulting oxidation product system is adjusted to 11-12, and after solid-liquid separation and washing, CaO2@FHMON nanoparticles are obtained;

[0042] Modifying the surface of the CaO2@FHMON nanoparticles with a biofilm-like coating to obtain CaO2@FHMON@Lip;

[0043] The CaO2@FHMON@Lip is dispersed in a polar organic solvent, and a NaHCO3 solution and a CD105 targeting ligand are sequentially added to the obtained dispersion. The CD105 targeting ligand is combined with the biomembrane biomimetic coating, and the nanocomplex is obtained after solid-liquid separation.

[0044] In the present invention, unless otherwise specified, all raw materials used are commercially available products well known in the art.

[0045] The preparation of FHMON is first described below.

[0046] The present invention mixes water, alcohol, ammonia water and a first silicate, performs a first hydrolysis and polycondensation, forms a silicon dioxide core, and obtains a core dispersion liquid.

[0047] In the present invention, the water is preferably deionized water, and the alcohol is preferably ethanol; the volume ratio of the water to the alcohol is preferably 1:7.

[0048] In the present invention, the concentration of the aqueous ammonia is preferably 0.1 to 2 M, and in specific embodiments, it can be 0.1 M, 0.5 M, 1 M, 1.5 M, or 2 M. In the present invention, the aqueous ammonia is used to provide an alkaline environment to promote the hydrolysis and polycondensation reactions of the first silicate. In the present invention, the volume ratio of water to aqueous ammonia is preferably 5:1.4 to 1.6. By controlling the concentration of the aqueous ammonia within this range, the present invention ensures a moderate hydrolysis rate, which facilitates the subsequent formation of a uniform and dense shell.

[0049] In the present invention, the first silicate preferably includes tetraethyl orthosilicate (TEOS); and the volume ratio of water to the first silicate is preferably 5:3.

[0050] In the present invention, the mixing of water, alcohol, aqueous ammonia and the first silicate preferably comprises: first mixing water, alcohol and aqueous ammonia, and then adding the first silicate to the resulting mixture.

[0051] The present invention has no special requirements for the mixing process of the water, alcohol and ammonia water, as long as the three can be mixed evenly. In the embodiment of the present invention, the stirring is continuously carried out at 30° C. for 30 minutes.

[0052] In the present invention, the first hydrolysis and polycondensation is preferably carried out under stirring conditions, and the reaction temperature of the first hydrolysis and polycondensation is preferably 25-35°C, and is 30°C in a specific embodiment. The present invention has no special requirements on the time of the first hydrolysis and polycondensation, as long as the first silicate is completely hydrolyzed and polycondensed, which is 45 minutes in a specific embodiment.

[0053] After obtaining the core dispersion, the present invention adds the mixture of perfluoroalkylsiloxane and the second silicate to the core layer dispersion for a second hydrolysis and polycondensation to form a fluorine-containing carbon chain-containing silica shell to cover the silica core, and collects colloidal nanoparticles with a core-shell structure.

[0054] In the present invention, the perfluoroalkylsiloxane preferably includes PDES; in the present invention, the type of the second silicate is preferably the same as the first silicate; the volume ratio of the perfluoroalkylsiloxane to the second silicate is preferably 1:2.5; and the volume ratio of the first silicate to the second silicate is preferably 3:2.5.

[0055] In the present invention, the temperature of the second hydrolysis and polycondensation is preferably 25-35°C, and in a specific embodiment, 30°C; the time of the second hydrolysis and polycondensation is preferably 70-90 minutes, and in a specific embodiment, 80 minutes. In the present invention, the mixture of the perfluoroalkylsiloxane and the second silicate is preferably slowly added dropwise to the core layer dispersion to prevent a violent reaction. In the present embodiment, the addition takes 3 minutes.

[0056] In the second hydrolysis and polycondensation process, the perfluoroalkylsiloxane and the second silicate undergo hydrolysis and polycondensation to form a hybrid shell layer, that is, a silica shell containing a fluorine-carbon chain is formed to cover the silica core.

[0057] After obtaining the core-shell colloidal nanoparticles, the present invention preferably rinses them with deionized water, and then removes the silica cores in the washed core-shell colloidal nanoparticles to obtain FHMON; the FHMON includes hollow mesoporous silica and carbon-fluorine chains doped in the hollow mesoporous silica shell skeleton.

[0058] The present invention has no special requirements for the method of removing the silica core, and a removal method well known in the art can be used. In the embodiment of the present invention, the silica core is dispersed in a 0.6M Na2CO3 aqueous solution, etched at 80°C for 30 minutes, and then centrifuged and washed with water to obtain FHMON.

[0059] After obtaining FHMON, the present invention mixes a water-soluble calcium salt, the FHMON, water, and a protective agent, adds an aqueous hydrogen peroxide solution to the resulting mixture for an oxidation reaction, in situ forming CaO2 within the cavity of the hollow mesoporous silica, adjusts the pH value of the resulting oxidation product system to 11-12, and obtains CaO2@FHMON nanoparticles after solid-liquid separation and washing.

[0060] In the present invention, the water-soluble calcium salt preferably includes CaCl2; the protective agent is preferably ammonia water and PEG-200; and the concentration of the ammonia water is preferably 1M.

[0061] In the present invention, the step of mixing the water-soluble calcium salt, the FHMON, water and the protective agent preferably comprises: firstly mixing the water-soluble calcium salt, FHMON and water, and then adding the protective agent to the obtained mixed liquid.

[0062] In the present invention, the mass ratio of the water-soluble calcium salt to FHMON is preferably 10:1; the concentration of the water-soluble calcium salt in the mixed liquid is preferably 0.1 g / mL. In the present invention, the volume ratio of the ammonia water to the mixed liquid is preferably 1:10; and the volume ratio of the PEG-200 to the mixed liquid is preferably 8:1.

[0063] In the present invention, the mass concentration of the aqueous hydrogen peroxide solution is preferably 30%; the volume ratio of the mass of the water-soluble calcium salt to the aqueous hydrogen peroxide solution is preferably 1 g:1 mL.

[0064] In the present invention, the aqueous hydrogen peroxide solution is preferably added dropwise. The rate of addition is not particularly limited, and dropwise addition is sufficient. In the embodiment of the present invention, one drop of aqueous hydrogen peroxide solution is added every 10 seconds.

[0065] In the present invention, the oxidation reaction is preferably carried out at room temperature (15-30° C.), and the oxidation reaction time is preferably 6 hours. In the oxidation reaction process of the present invention, hydrogen peroxide oxidizes the water-soluble calcium salt to CaO2.

[0066] In the present invention, the reagent used to adjust the pH of the resulting oxidation product system to 11-12 preferably includes a NaOH solution; the concentration of the NaOH solution is preferably 1 M. The pH value is adjusted in the present invention to maintain an alkaline environment. Calcium peroxide reacts with acid, and alkaline conditions, despite the presence of water, slow the consumption of calcium peroxide.

[0067] The present invention has no special requirements for the solid-liquid separation method, and any solid-liquid separation method well known in the art can be used, such as centrifugation.

[0068] In the present invention, the washing is preferably anhydrous ethanol washing.

[0069] In the present invention, the CaO2@FHMON nanoparticles include hollow mesoporous silica, carbon-fluorine chains doped in the shell skeleton of the hollow mesoporous silica, and CaO2 loaded in the cavity of the hollow mesoporous silica.

[0070] After obtaining the CaO2@FHMON nanoparticles, the present invention modifies the biomembrane biomimetic coating on the surface of the CaO2@FHMON nanoparticles.

[0071] Before modifying the biomembrane biomimetic coating, the present invention preferably disperses CaO2@FHMON nanoparticles and Toll-like receptor 7 / 8 dual agonist in a polar organic solvent, allowing the Toll-like receptor 7 / 8 dual agonist to enter the cavity of the hollow mesoporous silica, and performs solid-liquid separation to obtain Toll / CaO2@FHMON.

[0072] In the present invention, the polar organic solvent preferably includes dimethyl sulfoxide (DMSO). In the present invention, the mass ratio of the CaO2@FHMON nanoparticles to the Toll-like receptor 7 / 8 dual agonist is preferably 1:1. In the present invention, the volume ratio of the CaO2@FHMON nanoparticles to the polar organic solvent is preferably 10 mg:50 mL. After dispersing the CaO2@FHMON nanoparticles and the Toll-like receptor 7 / 8 dual agonist in the polar organic solvent, the present invention preferably continues stirring for 24 hours to allow the Toll-like receptor 7 / 8 dual agonist to enter the cavities of the hollow mesoporous silica.

[0073] In the present invention, the solid-liquid separation is preferably performed by centrifugation, the centrifugal force of the centrifugation is preferably 6000×g, and the centrifugation time is preferably 10 minutes. After the solid-liquid separation is completed, the present invention preferably washes the mixture three times with DMSO to remove residual Toll-like receptor 7 / 8 dual agonist and CaO2, thereby obtaining Toll / CaO2@FHMON.

[0074] In the present invention, the Toll / CaO2@FHMON comprises hollow mesoporous silica, a carbon-fluorine chain doped in the hollow mesoporous silica shell skeleton, and CaO2 and Toll-like receptor 7 / 8 dual agonist loaded in the hollow mesoporous silica cavity.

[0075] After obtaining Toll / CaO2@FHMON, the present invention modifies the surface of the Toll / CaO2@FHMON with a biomembrane biomimetic coating to obtain Toll / CaO2@FHMON@Lip.

[0076] The present invention does not require any special modification methods for the biomembrane biomimetic coating; modification methods well known in the art can be used. When the biomembrane biomimetic coating is a lipid layer, the modification method is as follows: R848 / CaO2@FHMON is dispersed in chloroform, DPPC and DSPE-PEG2000-NHS are added for ultrasonic dispersion, and then rotary evaporation is performed at 37°C for 30 minutes to form a lipid layer. The Toll / CaO2@FHMON@Lip is then separated by centrifugation at 8000×g for 10 minutes at room temperature.

[0077] In the present invention, the Toll / CaO2@FHMON@Lip includes hollow mesoporous silica, carbon fluorine chains doped in the shell skeleton of the hollow mesoporous silica, CaO2 and Toll-like receptor 7 / 8 dual agonist loaded in the cavity of the hollow mesoporous silica, and a biomembrane biomimetic coating modified on the surface of the hollow mesoporous silica.

[0078] After obtaining Toll / CaO2@FHMON@Lip, the present invention disperses the Toll / CaO2@FHMON@Lip in a polar organic solvent, sequentially adds a NaHCO3 solution and a CD105 targeting ligand to the resulting dispersion, combines the CD105 targeting ligand with the biomembrane biomimetic coating, and obtains the nanocomplex after solid-liquid separation.

[0079] In the present invention, the polar organic solvent preferably includes chloroform, and the concentration of Toll / CaO2@FHMON@Lip in the dispersion is preferably 2 mg / mL. In the present invention, the concentration of the NaHCO3 solution is preferably 0.1 M; the volume ratio of the NaHCO3 solution to the polar organic solvent is preferably 1:1. In the present invention, after adding the CD105 targeting ligand, the mixture is preferably stirred for 4 hours in the dark. In the present invention, when the biomembrane biomimetic coating is a lipid layer, the CD105 targeting ligand specifically binds to amino groups in the lipid layer.

[0080] In the present invention, the solid-liquid separation method is preferably centrifugation, and in the embodiment of the present invention, it is specifically high-speed centrifugation at 8000×g for 10 minutes at room temperature.

[0081] The present invention provides the use of the nanocomplex described in the above scheme in the preparation of anti-tumor products.

[0082] Prior to application, the nanocomplex is preferably subjected to oxygen adsorption. In an embodiment of the present invention, the nanocomplex is placed in deionized water, oxygen is then bubbled into the deionized water, and after oxygen adsorption, solid-liquid separation is performed to obtain the oxygen-adsorbed nanocomplex.

[0083] The nanocomplex provided by the present invention, its preparation method and application are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0084] Example 1

[0085] Figure 1 Schematic diagram of the preparation of the AL@O2-FMRC nanocomplex in Example 1 of the present invention.

[0086] (1) Synthesis of FHMON (FM)

[0087] 5 mL of deionized water and ethanol were mixed in a 1:7 volume ratio, and 1.57 mL of 1 M ammonia was added to a round-bottom flask. Stirring was continued at 30°C for 30 minutes. 3 mL of TEOS was added, and stirring was continued for an additional 45 minutes to allow complete hydrolysis and polycondensation of TEOS, forming a silica core (s-SiO2). Subsequently, a mixture of TEOS (2.5 mL) and PDES (1 mL) was dripped into the milky white dispersion over 3 minutes. Incubation was continued at 30°C for an additional 80 minutes to coat the silica core with an organosilicon hybrid shell, resulting in core-shell colloidal nanoparticles (abbreviated as s-SiO2@h-SiO2). The white colloidal nanoparticles were then collected, rinsed with deionized water, and divided equally into two portions. In 50 mL of Na2CO3 aqueous solution (0.6 M), it was dispersed at 80°C for another 30 min. During this process, a solid core (i.e., s-SiO2) was etched out. After high-speed centrifugation at 12000 rpm / min for 10 min, it was washed three times with deionized water to obtain the final product, i.e., hollow mesoporous silica nanoparticles doped with fluorocarbon chains in the shell, denoted as FHMON, further abbreviated as FM.

[0088] (2) In situ CaO2 synthesis on FHMON carrier (CaO2@FHMON, i.e. FMC)

[0089] A 10 mL aqueous solution of CaCl2 (0.1 g / mL) and FHMONs (100 mg) was placed in a flask. 1 mL of 1 M ammonia solution and 80 mL of PEG-200 solution were added. The mixture was rapidly stirred while 1 mL of 30% H2O2 solution was slowly added (one drop every 10 seconds). Stirring was continued at room temperature for 6 hours to obtain a colorless, transparent solution. Under ultrasonication, a 1 M NaOH solution was slowly added to adjust the pH to 11.5. The mixture was then centrifuged at 10,000 rpm for 10 minutes to obtain a white precipitate, which was then washed with anhydrous ethanol to yield white CaO2@FHMON nanoparticles.

[0090] (3) Synthesis of R848 / CaO2@FHMON(FMRC)

[0091] 10 mg of R848 (purchased from Meilun Bio) and 10 mg of CaO2@FHMON were dispersed in 50 mL of DMSO using ultrasound-assisted dispersion. Stirring was then continued for 24 h to achieve efficient loading of R848 by CaO2@FHMON. The mixture was centrifuged at 6000 × g for 10 min at room temperature and washed three times with DMSO to remove residual R848 and CaO2, yielding the R848 / CaO2@FHMON product. To verify the successful loading of R848 and CaO2, UV-visible spectroscopy was performed on a Nicolet Avatar 370 FT-IR spectrophotometer. The results are shown in Table 1. Figure 2 .Depend on Figure 2 It can be seen that FMRC has an obvious UV absorption peak compared with FM, indicating that R848 is successfully loaded into FM.

[0092] (4) Synthesis of R848 / CaO2@FHMON@Lip(L@FMRC)

[0093] 10 mg of R848 / CaO2@FHMON was dispersed in 5 mL of chloroform. 16 mg of DPPC and 28 mg of DSPE-PEG2000-NHS were then added and ultrasonically dispersed throughout the R848 / CaO2@FHMON chloroform solution. The mixture was then rotated and evaporated at 37°C for 30 minutes. The R848 / CaO2@FHMON@Lip product was obtained by high-speed centrifugation at 8000 × g for 10 minutes at room temperature.

[0094] (5) Synthesis of R848 / CaO2@FHMON@Lip-antiCD105(AL@FMRC) and R848 / CaO2@O2-FHMON@Lip-antiCD105(AL@O2-FMRC)

[0095] 10 mg of R848 / CaO2@FHMON@Lip was dispersed in 5 mL of chloroform, 5 mL of NaHCO3 (0.1 M) solution was added, and then 10 μL of anti-CD105 monoclonal antibody at a concentration of 1 mg / mL (purchased from Abcam, the trade name is recombinant Anti-CD105 antibody [epr19911-220] (ab252345)) was added. The mixture was stirred at room temperature in the dark for 4 h and centrifuged at 8000 × g for 10 min at room temperature to obtain R848 / CaO2@FHMON@Lip-antiCD105.

[0096] R848 / CaO2@FHMON@Lip-antiCD105 was added to deionized water and O2 was bubbled for 1 h to obtain R848 / CaO2@O2-FHMON@Lip-antiCD105.

[0097] The zeta potential and particle size distribution of FM, FMRC, L@FMRC and AL@FMRC were investigated on MalvernNano-ZS90. Figure 3 .Depend on Figure 3 It can be seen that the FM potential increased after adding calcium peroxide and R848, indicating that the loading was successful. After coating with liposomes, the potential decreased again, indicating that the liposomes successfully encapsulated FMRC. After antiCD105 was coupled to the liposome surface, the potential increased again, indicating that antiCD105 was successfully coupled to the liposomes.

[0098] Figure 4 In the first row, from left to right, the first picture is the structural diagram of AL@FMRC, the second picture is the transmission electron microscope picture of AL@FMRC, the third picture is the HAADF-STEM picture of AL@FMRC, the fourth picture in the first row is the element mapping of AL@FMRC, and the pictures in the second and third rows are the element mapping pictures. Figure 4 It can be seen that C, N, Si, F, O, S, P and Ca elements are distributed in AL@FMRC, indicating that R848 and CaO2 are successfully loaded. Figure 4 It can also be seen that the cavity diameter of the hollow mesoporous silica is approximately 200 nm.

[0099] Example 2

[0100] CaO2@FHMON@Lip-antiCD105(AL@FMC)

[0101] The only difference from Example 1 is that step (3) is omitted, that is, R848 is not loaded.

[0102] Test Example 1: Oxygen Generation Capacity Test

[0103] AL@O2-FMC, AL@FM, AL@O2-FMR and AL@O2-FMRC were tested for their oxygen generation capabilities.

[0104] The preparation method of AL@O2-FMC is as follows: the AL@FMC of Example 2 is placed in deionized water and O2 is bubbled for 1 hour to obtain AL@O2-FMC.

[0105] The preparation of AL@FM is the same as that in Example 1, except that steps (2) and (3) are omitted, i.e., CaO2 and R848 are not loaded.

[0106] The preparation method of AL@O2-FMR is as follows: on the basis of Example 1, step (2) is omitted, that is, CaO2 is not loaded, AL@FMR is first prepared, and then AL@FMR is placed in deionized water and O2 is bubbled for 1 hour to obtain AL@O2-FMR;

[0107] The blank control group (Control) consisted of deionized water without any treatment.

[0108] The oxygen generation capacity test steps are as follows:

[0109] The above groups were placed under ultrasound conditions (ultrasound conditions: ultrasound 30s, 1MHz, 0.5W / cm 2 ) to make a smear and observe whether there are bubbles under a microscope. Figure 5 .Depend on Figure 5It can be seen that no bubbles were generated in each group without ultrasound; after ultrasound, bubbles were generated in AL@O2-FMR, AL@O2-FMC and AL@O2-FMRC, indicating that the carried oxygen was successfully released.

[0110] Figure 6 Schematic diagram of oxygen generation in AL@O2-FMRC. The oxygen carried by the fluorocarbon chain is released under ultrasound conditions, thereby alleviating tumor hypoxia.

[0111] Test Example 2: Immune Regulation Ability Test

[0112] The nanocomplex AL@FMRC was co-incubated with M2 macrophages for 3 hours, and the expression of interleukin-10, a marker of M2 macrophages, was detected by ELISA. Similarly, the nanocomplex without R848 (i.e., AL@FMC in Example 2) was co-cultured with M2 macrophages to detect the expression of interleukin-10, a marker of M2 macrophages. The results are shown in Figure 2. Figure 8 As shown, compared with the nanocomplex AL@FMC without R848 loading, the expression level of interleukin-10 in the nanocomplex AL@FMRC group loaded with R848 was significantly reduced, indicating that R848 significantly promoted the transformation of M2 macrophages to M1 macrophages.

[0113] Figure 7 These are morphological images of macrophages of different forms under a microscope, used to illustrate that different macrophages have different morphologies, and M2 cells are more likely to transform into M1 cells.

[0114] Test Example 3: Tumor Targeting Ability Test

[0115] The nano-modulator was prepared into an injection solution with a concentration of 200 μg / ml (the solvent was PBS solution), and 200 μL of the injection solution was injected into the tumor-bearing mice through the tail vein. The accumulation of the nano-modulator in the tumor site was observed by fluorescence imaging. The results are shown in Figure 9 .Depend on Figure 9 It can be seen that the accumulation of nano-regulators in the tumor site is significantly higher than that in other tissues.

[0116] Referring to the above method, the unmodified anti-CD105 nanocomplex (i.e., L@FMRC) was injected into tumor-bearing mice, and its accumulation in the tumor site was observed by fluorescence imaging. Figure 9 .Depend on Figure 9 It can be seen that the accumulation amount of the unmodified anti-CD105 nanocomplex at the tumor site is significantly lower than that of the modified anti-CD105 nanomodulator.

[0117] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A nanocomplex, characterized in that: The invention comprises hollow mesoporous silica; the interior of the hollow mesoporous silica is a cavity, and the exterior is a shell with a mesoporous structure; the nanocomplex also comprises a carbon fluorine chain doped in the hollow mesoporous silica shell skeleton, CaO2 loaded in the hollow mesoporous silica cavity, and a CD105 targeting ligand modified on the surface of the hollow mesoporous silica by a biomembrane biomimetic coating; the carbon fluorine chain is formed by hydrolysis of perfluoroalkylsiloxane.

2. The nanocomplex according to claim 1, characterized in that The cavity of the hollow mesoporous silica also includes a Toll-like receptor 7 / 8 dual agonist.

3. The nanocomplex according to claim 2, characterized in that The Toll-like receptor 7 / 8 dual agonist includes resiquimod.

4. The nanocomplex according to claim 1, characterized in that The biomembrane biomimetic coating comprises a lipid layer, a cell membrane or red blood cells.

5. The nanocomplex according to claim 1, characterized in that The CD105 targeting ligand includes an anti-CD105 monoclonal antibody or a nucleic acid aptamer.

6. The nanocomplex according to claim 1, characterized in that The perfluoroalkylsiloxane includes 1H,1H,2H,2H-perfluorodecyltriethoxysilane.

7. The nanocomplex according to claim 1, characterized in that The pore diameter of the hollow mesoporous silica is 2-10 nm, the cavity diameter of the hollow mesoporous silica is 50-300 nm, and the shell thickness is 10-30 nm.

8. The method for preparing the nanocomplex according to any one of claims 1 to 7, characterized in that: The following steps are involved: Mixing water, alcohol, ammonia water and a first silicate, performing a first hydrolysis and polycondensation to form a silica core, and obtaining a core dispersion; adding a mixture of perfluoroalkylsiloxane and a second silicate to the core layer dispersion, performing a second hydrolysis and polycondensation to form a silica shell containing a fluorine-carbon chain to coat the silica core, and collecting colloidal nanoparticles having a core-shell structure; removing the silica core from the colloidal nanoparticles having the core-shell structure to obtain FHMON; the FHMON comprises hollow mesoporous silica and carbon-fluorine chains doped in the hollow mesoporous silica shell skeleton; A water-soluble calcium salt, the FHMON, water and a protective agent are mixed, and an aqueous hydrogen peroxide solution is added to the resulting mixture to carry out an oxidation reaction, thereby in situ forming CaO2 in the cavity of the hollow mesoporous silica. The pH value of the resulting oxidation product system is adjusted to 11-12, and after solid-liquid separation and washing, CaO2@FHMON nanoparticles are obtained; Modifying the surface of the CaO2@FHMON nanoparticles with a biofilm-like coating to obtain CaO2@FHMON@Lip; The CaO2@FHMON@Lip is dispersed in a polar organic solvent, and a NaHCO3 solution and a CD105 targeting ligand are sequentially added to the obtained dispersion. The CD105 targeting ligand is combined with the biomembrane biomimetic coating, and the nanocomplex is obtained after solid-liquid separation.

9. The preparation method according to claim 8, characterized in that When the cavity of the hollow mesoporous silica also includes a Toll-like receptor 7 / 8 dual agonist, the method further includes: dispersing CaO2@FHMON nanoparticles and the Toll-like receptor 7 / 8 dual agonist in a polar organic solvent, allowing the Toll-like receptor 7 / 8 dual agonist to enter the cavity of the hollow mesoporous silica, performing solid-liquid separation to obtain Toll / CaO2@FHMON, and then modifying the surface of the Toll / CaO2@FHMON with a biomembrane biomimetic coating.

10. Use of the nanocomplex according to any one of claims 1 to 7 or the nanocomplex prepared by the preparation method according to any one of claims 8 to 9 in the preparation of anti-tumor products.

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