Protein nanoparticle with deformability and dual-targeting capability in response to pH as well as preparation method and application of protein nanoparticle

Abstract

The invention discloses a pH-responsive deformable dual-targeting nano protein and a preparation method and application thereof, the method comprises the following steps: activating carboxylated mannose through an EDC / NHS system, reacting with an albumin solution, and dialyzing and purifying to obtain mannose modified albumin; and mixing the mannose modified albumin with an inhibitor IRG1-IN-1, and loading the IRG1-IN-1 through a hydrophobic effect to obtain the mannose albumin nanoparticles, ferritin serves as a carrier, oxaliplatin is loaded through a solvent replacement method, and ferritin nanoparticles are obtained; cHO-PEG2k-CHO is used as a coupling agent, and the mannose albumin nanoparticles and the ferritin nanoparticles are crosslinked through a Schiff base reaction, so that the large-size pH-responsive dual-targeting nano-protein is formed. The nano protein has the characteristic of double-targeting separability, so that specific treatment can be realized, immune cell metabolism is interfered while tumor cells are killed, metabolic immunosuppression is blocked, and anti-tumor immunotherapy is promoted.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a pH-responsive protein nanoparticle having deformability and dual-targeting capabilities, and a preparation method and application thereof. Background Art

[0002] With the continuous development of tumor treatment strategies, traditional treatment methods such as surgery, radiotherapy and chemotherapy still occupy the mainstream position, but these methods often ignore the complexity of immune cell metabolism in the tumor immune microenvironment. The immune escape mechanism of tumors is not only closely related to the mutation and drug resistance of tumor cells themselves, but also closely related to the abnormal metabolism of immune cells in the tumor microenvironment.

[0003] Studies have shown that the hypoxic, acidic environment and high metabolic demand of the tumor microenvironment greatly affect the function of immune cells. In particular, the metabolic changes of important immune cells such as macrophages and T cells in the tumor microenvironment inhibit their normal immune response ability, thereby leading to the failure of anti-tumor immunity and poor prognosis. For example, the metabolism of tumor cells changes the metabolic pathways of macrophages, promotes the transformation of macrophages to an immunosuppressive phenotype, and inhibits the ability of macrophages to recognize and eliminate tumors. In addition, the activity of immunosuppressive cells such as tumor-associated macrophages and regulatory T cells also enhances the immune escape function of tumors through metabolic changes. The metabolic disorders of these immune cells are closely related to the growth and metastasis of tumors, which directly leads to unsatisfactory tumor treatment effects and poor patient prognosis.

[0004] Therefore, it is necessary and urgent to develop a multifunctional dual-targeted nanomaterial that can specifically respond to the tumor microenvironment, kill tumor cells, and regulate the metabolism of immune cells. Summary of the invention

[0005] The purpose of the present invention is to provide a pH-responsive protein nanoparticle with deformability and dual-targeting ability, as well as a preparation method and application thereof. The pH-responsive deformation ability makes it highly specific, and it can actively target the tumor microenvironment and can target macrophages and tumor cells respectively after deformation, thereby improving the high efficiency of treatment, compensating for the immunosuppressive microenvironment caused by single targeted tumor treatment, thereby comprehensively improving the high efficiency of treatment and realizing the ingenious fusion of chemotherapy and immunotherapy.

[0006] In a first aspect of the present invention, a protein nanoparticle having pH-responsive deformability and dual-targeting capability and a preparation method thereof are provided, the method comprising:

[0007] After the carboxylated mannose was activated by the EDC / NHS system, it was reacted with the albumin solution and purified by dialysis to obtain the mannose-modified albumin.

[0008] The mannose-modified albumin is mixed with the inhibitor IRG1-IN-1, and the IRG1-IN-1 is loaded by hydrophobic interaction to prepare mannose albumin nanoparticles;

[0009] Ferritin nanoparticles were prepared by using ferritin as a carrier and loading oxaliplatin via solvent replacement method.

[0010] Using CHO-PEG2k-CHO as a linker, the mannose albumin nanoparticles and the ferritin nanoparticles are cross-linked through a Schiff base reaction to form a large-sized pH-responsive dual-targeting nanoprotein.

[0011] Furthermore, the specific conditions of the EDC / NHS activation system are:

[0012] The molar ratio of EDC, NHS and carboxylated mannose is (1.5-2.5): (1.5-2.5): 1, the activation reaction time is 6-10 hours, and the reaction temperature is 20-25°C.

[0013] Furthermore, the concentration of the carboxylated mannose is 0.5-2 mM, and the mass ratio of albumin to carboxylated mannose is 1:(0.1-0.5).

[0014] Further, the method of loading IRG1-IN-1 includes:

[0015] After IRG1-IN-1 was dissolved in ethanol, it was added dropwise to the mannose albumin solution containing β-mercaptoethanol at a rate of 5-10 μL / s, stirred for reaction for 4-8 hours, and then centrifuged for purification.

[0016] Furthermore, the conditions for loading oxaliplatin are:

[0017] The concentration of ferritin is 1-3 mg / mL, the molar ratio of oxaliplatin to ferritin is (5-20):1, and the solvent is a mixed system of DMF and ultrapure water, wherein the volume ratio of DMF to ultrapure water is 1:10-1:20.

[0018] Further, the conditions of the Schiff base reaction include:

[0019] The reaction pH is 8-10, the mass ratio of albumin nanoparticles to ferritin nanoparticles is (3:1)-(5:1), and the reaction time is 4-8 hours.

[0020] Furthermore, the added mass of the CHO-PEG2k-CHO is 5%-15% of the total mass of albumin and ferritin.

[0021] In a second aspect of the present invention, a pH-responsive deformable dual-targeting nanoprotein prepared by the method is provided.

[0022] (a) Maintaining large-size structures of 50-70 nm at pH ≥ 7.4;

[0023] (b) dissociated into 10-15 nm mannose albumin nanoparticles and ferritin nanoparticles at pH ≤ 6.5;

[0024] (c) Mannosalbumin nanoparticles target macrophages and inhibit the IRG1-ITA pathway, while ferritin nanoparticles target tumor cells and induce DNA damage.

[0025] Furthermore, the mannose modification degree of the mannose albumin nanoparticles is 3-8 mannose molecules connected to each albumin molecule;

[0026] The oxaliplatin loading amount of the ferritin nanoparticles is 8%-15% (w / w), and the encapsulation rate of IRG1-IN-1 in the mannose albumin nanoparticles is ≥85%.

[0027] In the third aspect of the present invention, the use of the dual-targeting nanoprotein in the preparation of anti-tumor drugs is provided.

[0028] (i) Tumor microenvironment-specific dual targeting via pH-responsive dissociation;

[0029] (ii) synchronously kill tumor cells and regulate macrophage metabolism to relieve immunosuppression;

[0030] (iii) Activate the STING pathway and promote the secretion of inflammatory factors TNF-α, IL-6, and IFN-β.

[0031] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0032] 1. The present invention provides a pH-responsive protein nanoparticle with deformability and dual-targeting ability, which has the ability to actively target tumors and can respond to the slightly acidic environment of tumors, deforming from large-sized protein nanoparticles to small-sized protein nanoparticles, targeting tumor cells and macrophages respectively, and has strong specificity for precise cell regulation.

[0033] 2. The protein nanoparticles provided by the present invention have pH-responsive deformability and dual-targeting capabilities. In the process of tumor treatment, by responding to the acidic pH in the tumor microenvironment, a series of cascade reactions are initiated, including:

[0034] Through CHO-PEG 2kAfter the large-sized protein nanoparticles formed by the -CHO connection reach the slightly acidic environment of the tumor, the Schiff base breaks, releasing small-sized protein nanoparticles that target tumors and macrophages respectively. There are transferrin receptors on ferritin, which actively targets tumors and is internalized by tumor cells, releasing the chemotherapy drug oxaliplatin, promoting tumor cell DNA damage, tumor cell necrosis, and releasing mtDNA; while the albumin nanoparticles with mannose can actively target macrophages with mannose receptors and be internalized, releasing IRG1-IN-1 drugs, interfering with the IRG1-ITA pathway, downregulating the expression of IRG1, and thus reducing the production of itaconic acid (ITA), an immunosuppressive metabolite of macrophages. The mtDNA released by tumor cell death promotes the activation of macrophage STING, and the reduction of itaconic acid content further slows down the inhibition of the STING pathway, further promoting the activation of STING, and promoting the occurrence of anti-tumor immunity.

[0035] 3. The protein nanoparticles provided by the present invention are pH-responsive, deformable and dual-targeting. In the process of tumor treatment, they not only kill tumor cells, but also regulate the metabolism of macrophages, reduce the production of immunosuppressive metabolites such as itaconic acid, promote the maturation of dendritic cells, induce macrophage polarization from immunosuppressive M2 to immune-activated M1, activate the killing function of effector T cells, and enhance the anti-tumor immune response.

[0036] 4. The present invention provides a pH-responsive protein nanoparticle with deformability and dual-targeting ability and a preparation method thereof. The pH-responsive deformation promotes the occurrence of precise dual-targeting, avoids the limitations of single tumor cell regulation or single immune cell regulation, and avoids the defect of insufficient targeting due to simultaneous input of dual-targeting proteins.

[0037] 5. The present invention provides a pH-responsive protein nanoparticle with deformability and dual-targeting ability and a preparation method thereof. The preparation is simple and convenient, and the prepared protein nanoparticles are stable in nature and have efficient and accurate tumor treatment effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0039] Figure 1 To construct pH-responsive protein nanoparticles with deformability and dual targeting capabilities (IRMH-CHO-OFe) and their anti-tumor mechanisms;

[0040] Figure 2 TEM images of small-sized ferritin nanoparticles (Oxa@Fep), small-sized albumin nanoparticles (IRG1-IN-1@MHSA), and large-sized pH-responsive dual-targeted protein nanoparticles (IRMH-CHO-OFe); wherein, Figure 2 A is a TEM image of small-sized albumin nanoparticles loaded with IRG1-IN-1. Figure 2 B is a TEM image of small-sized ferritin nanoparticles loaded with oxaliplatin. Figure 2 C is CHO-PEG 2k -TEM image of large-sized protein nanoparticles formed by CHO connection;

[0041] Figure 3 CHO-PEG 2k -CHO-linked large protein nanoparticles and MAL-PEG 2k -TEM image of large-sized protein nanoparticles formed by MAL connection;

[0042] Figure 4 To characterize the in vitro tumor toxicity of protein nanoparticles with pH-responsive deformability and dual-targeting capabilities; Figure 4 A is the toxicity results of pH-responsive protein nanoparticles with deformability and dual-targeting ability to CT26 tumor cells at different material concentrations under pH 7.4. Figure 4 B is the toxicity results of pH-responsive protein nanoparticles with deformability and dual-targeting ability to CT26 tumor cells at different material concentrations under pH 6.5. Figure 4 C shows the toxicity results of different groups of materials on CT26 tumor cells, including blank control group (control), small-sized albumin nanoparticles (IRG1-IN-1@MHSA), small-sized ferritin nanoparticles (Oxa@Fep) and MAL-PEG 2k -MAL-linked large protein nanoparticles (IRMH-MAL-OFe) and CHO-PEG 2k - Large-sized protein nanoparticles formed by CHO connection (IRMH-CHO-OFe);

[0043] Figure 5 To characterize the tumor cell killing results of protein nanoparticles with pH-responsive deformability and dual-targeting ability; Figure 5 A is the comet assay to evaluate the DNA damage of CT26 cells by different materials after incubation with CT26 tumor cells. Figure 5B is the immunofluorescence measurement of the degree of DNA damage of CT26 induced by different materials after incubation of CT26 tumor cells with different materials. Figure 5 C is the quantitative evaluation of DNA damage of CT26 tumor cells by different materials. Figure 5 D is the supernatant of different materials incubated with CT26 and then incubated with macrophages to measure the content of inflammatory factor TNF-α produced in the supernatant of macrophages. Figure 5 E is the supernatant of different materials incubated with CT26 and then incubated with macrophages to measure the content of inflammatory factor IL-6 produced in the supernatant of macrophages. Figure 5 F: The supernatants of different materials incubated with CT26 were then incubated with macrophages to measure the content of IFN-β produced in the supernatants of macrophages;

[0044] Figure 6 A flow chart of a method for preparing pH-responsive protein nanoparticles having deformability and dual-targeting capabilities provided in an embodiment of the present invention.

[0045] Figure 7 This is the TEM image of the nanoparticles prepared in Comparative Example 2. DETAILED DESCRIPTION

[0046] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0047] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0048] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0049] Ferritin (derived from horse spleen): product number F4503-100MG, purchased from Sigma-Aldrich.

[0050] Human serum albumin: Product number S12018-1g, purchased from Yuanye.

[0051] CHO-PEG 2k-CHO, name: aldehyde polyethylene glycol aldehyde aldehyde PEG aldehyde, alias: dialdehyde polyethylene glycol, English name: CHO-PEG-CHO ALD-PEG-ALD, molecular weight (PEG) is 2000, purchased from Chongqing Yusi Pharmaceutical.

[0052] The present invention provides a protein nanoparticle having pH-responsive deformability and dual-targeting capability, such as Figure 6 As shown, the overall idea is as follows:

[0053] According to another typical embodiment of the present invention, a method for preparing protein nanoparticles having pH-responsive deformability and dual-targeting capabilities is provided, such as Figure 6 As shown, the method includes:

[0054] S1, activating carboxylated mannose and modifying it on albumin, and obtaining mannose-modified albumin after dialysis and freeze-drying;

[0055] In step S1, by changing the concentration of activated mannose added, small-sized albumin nanoparticles with different degrees of mannose connection can be obtained.

[0056] As an optional implementation, the step S1 specifically includes:

[0057] Completely dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in an ultrapure aqueous solution at an equal molar ratio, then adding a carboxymannose aqueous solution, stirring at room temperature for 8 hours to obtain carboxyl-activated mannose, further, modifying albumin after activating the carboxymannose, dissolving a certain amount of albumin in ultrapure water and ultrasonically dispersing it, then adding the albumin solution to the carboxyl-activated carboxymannose solution, stirring and dialyzing for 2 days, and lyophilizing to obtain mannose-modified albumin;

[0058] Wherein, the molar ratio of EDC, NHS and carboxylated mannose is (1.5-2.5): (1.5-2.5): 1, preferably, the molar ratio of EDC, NHS and carboxylated mannose is 2:2:1;

[0059] The concentration of the hydroxymannose aqueous solution may be 0.5-2 mM;

[0060] The stirring time of adding the albumin solution to the carboxyl-activated carboxyl mannose solution is 24-48 hours;

[0061] S2, dissolving mannose-modified albumin in water, adding inhibitor IRG1-IN-1 and stirring, thereby obtaining small-sized mannose-modified albumin nanoparticles;

[0062] The step S2 specifically includes:

[0063] A certain amount of IRG1-IN-1 was dissolved in ethanol and ultrasonically dispersed. 5 mg of mannose-modified albumin was dissolved in 5 ml of ultrapure water. After ultrasonic dispersion, 35 ul of β-mercaptoethanol was added. After stirring for 2 minutes, the IRG1-IN-1 dissolved in ethanol was slowly added dropwise to the albumin solution. After stirring for 6 hours, the solution was centrifuged to obtain small-sized albumin nanoparticles loaded with IRG1-IN-1.

[0064] The solvent of the IRG1-IN-1 solution is ethanol, and the dripping speed is controlled at 5-10 μL / s. The nanoparticles formed at a rate within this range are relatively uniform. If the dripping speed is less than 5 μL / s or greater than 10 μL / s, the formed nanoparticles are easily irregular, which is not conducive to the subsequent protein-protein connection reaction.

[0065] S3, using ferritin as a carrier to load the chemotherapy drug oxaliplatin to obtain small-sized ferritin nanoparticles;

[0066] The concentration of ferritin is 1-3 mg / mL, the molar ratio of oxaliplatin to ferritin is 1:(2-10), the solvent is a mixed system of DMF and ultrapure water, and the volume ratio of DMF to ultrapure water is 1:10-1:20.

[0067] As a specific implementation, step S3 specifically includes:

[0068] A certain amount of oxaliplatin was dissolved in DMF and ultrasonically dispersed, ferritin (2 mg / mL, 500 ul) was added to 10 ml of ultrapure water and ultrasonically dispersed, and then oxaliplatin dissolved in DMF was added, and stirred at room temperature for 12 h to obtain small-sized ferritin nanoparticles loaded with oxaliplatin;

[0069] S4. Add CHO-PEG 2k -CHO was used as a linker to connect small-sized albumin nanoparticles and ferritin nanoparticles through Schiff base reaction to obtain large-sized dual-targeted nanoproteins that respond to pH deformation;

[0070] The conditions of the Schiff base reaction include:

[0071] The mass ratio of albumin nanoparticles to ferritin nanoparticles is (3:1)-(5:1). Preferably, the mass ratio of the small-sized albumin to the small-sized ferritin is 5:1.

[0072] The reaction pH is 8-10 (preferably 8.2), and the reaction time is 4-8 hours.

[0073] The step S4 specifically includes:

[0074] The small-sized albumin nanoparticles and ferritin nanoparticles obtained above were added to 5 ml of ultrapure water, and then 5 ul-15 ul (preferably 10 ul) of NaOH (1 M) was added. After stirring for 2 min, ethanol solution was added and stirred for 2-4 h, and then the protein linker CHO-PEG was added. 2k -CHO, react for 5-7 hours and then wash with a 10W ultrafiltration tube to obtain a pH-responsive and deformable dual-targeting nanoprotein.

[0075] The reason for adding 5ul-15ul (preferably 10ul) NaOH (1M) is to adjust the pH of the solution. 2k -CHO connects two proteins through a Schiff base reaction, and under alkaline conditions (pH 8-10), the Schiff base reaction can proceed more stably, and the two proteins are more easily connected into large-sized pH-responsive dual-targeted protein nanoparticles;

[0076] According to another typical embodiment of the present invention, provided are protein nanoparticles prepared by the method and having pH-responsive deformability and dual-targeting capabilities.

[0077] According to another typical embodiment of the present invention, there is provided the use of the pH-responsive protein nanoparticles having deformability and dual-targeting capabilities as anti-tumor drugs.

[0078] This pH-responsive protein nanoparticle with deformability and dual-targeting capabilities can actively cleave into two functionally independent subunits in the micro-acidic environment of the tumor, targeting tumor cells and macrophages respectively. This "environmental trigger-functional typing" mechanism breaks through the limitation of traditional single-targeted or dual-targeted carriers that rely only on a single response to release drugs, and the deformation mechanism and dual targeting synergistically enhance the effect, which can not only kill tumor cells, but also accurately regulate macrophage metabolism, downregulate the expression of macrophage IRG1 gene, thereby downregulating the production of immunosuppressive metabolite itaconic acid, unblocking the inhibitory effect of itaconic acid on other immune cells, and promoting the occurrence of anti-tumor immunity. In addition, the nanoparticle cleavage is achieved through pH-sensitive imine bonds, responding to the micro-acidic environment of the tumor. The deformed small-sized subunits are easier to penetrate the dense tumor matrix, further improving their penetration depth, solving the problem of traditional nanoparticles being limited by the EPR effect. In addition, natural protein carriers have low immunogenicity, are easier to be cleared from the body, reduce the risk of long-term toxicity, and have strong biosafety. Figure 1 As shown, the design and development of this pH-responsive deformable and dual-targeting protein nanoparticle provides a new strategy for the efficient treatment of tumors and a new idea for the clever combination of chemotherapy and immunotherapy.

[0079] The following is a detailed description of a pH-responsive protein nanoparticle with deformability and dual-targeting capabilities of the present application in combination with examples and experimental data.

[0080] Example 1. pH-responsive deformable dual-targeting nanoprotein and preparation method thereof

[0081] 1. Mannose-modified albumin and preparation method thereof

[0082] The 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in an equal molar ratio are completely dissolved in an ultrapure aqueous solution, wherein the molar ratio of EDC, NHS and carboxymannose is 2:2:1, and then the carboxymannose aqueous solution is added, and stirred at room temperature for 8 hours to obtain a carboxyl-activated mannose solution. Further, 10 mg of albumin is dissolved in ultrapure water for ultrasonic dispersion (the mass volume ratio of the albumin to the ultrapure water is 1:1), and then the albumin solution is added to the carboxyl-activated carboxylmannose solution (the volume ratio of the albumin solution to the carboxyl-activated carboxylmannose solution is 2:1), the concentration of the hydroxymannose aqueous solution can be 0.5 mM, and after stirring for 24 hours, it is dialyzed for 2 days and freeze-dried to obtain mannose-modified albumin.

[0083] Dissolve 1 mg of IRG1-IN-1 in 500 ul of ethanol and disperse it ultrasonically. Take 5 mg of mannose-modified albumin and dissolve it in 5 ml of ultrapure water. After ultrasonic dispersion, add 35 ul of β-mercaptoethanol. After stirring for 2 minutes, slowly add the IRG1-IN-1 dissolved in ethanol to the albumin solution. After stirring for 6 hours, centrifuge to obtain small-sized albumin nanoparticles loaded with IRG1-IN-1.

[0084] 2. Small-sized albumin nanoparticles loaded with IRG1-IN-1 and preparation methods thereof

[0085] 1 mg of IRG1-IN-1 (the inhibitor IRG1-IN-1 of the present invention was purchased from MCE with the item number HY-148335) was dissolved in 500 ul of ethanol and ultrasonically dispersed. 5 mg of mannose-modified albumin was dissolved in 5 ml of ultrapure water. After ultrasonic dispersion, 35 ul of β-mercaptoethanol was added. After stirring for 2 min, the IRG1-IN-1 dissolved in ethanol was slowly added dropwise to the albumin solution (dropping rate was 5-10 μL / s). After stirring for 6 h, the small-sized albumin nanoparticles loaded with IRG1-IN-1 were obtained by centrifugation.

[0086] 3. Small-sized ferritin nanoparticles loaded with oxaliplatin and preparation method thereof

[0087] 1 mg of oxaliplatin was dissolved in 500 ul of DMF and ultrasonically dispersed to obtain a DMF solution containing oxaliplatin. Ferritin (2 mg / mL, 500 ul) was added to 10 ml of ultrapure water and ultrasonically dispersed. Then, the DMF solution containing oxaliplatin was added and stirred at room temperature for 12 h to obtain small-sized ferritin nanoparticles loaded with oxaliplatin.

[0088] 4. Protein nanoparticles with pH-responsive deformability and dual-targeting capabilities and preparation methods thereof

[0089] The small-sized albumin nanoparticles and ferritin nanoparticles obtained above were added to 5 ml of ultrapure water at a mass ratio of 5:1, and then 10 ul NaOH (1 M) was added to react to a pH of 8.2. After stirring for 2 minutes, ethanol solution was added and stirred for 3 hours. Then 200 ul (100 mM) of protein linker CHO-PEG2k-CHO was added. After reacting for 6 hours, centrifugal washing with a 10w ultrafiltration tube was performed to obtain a pH-responsive deformable dual-targeted nanoprotein (IRMH-CHO-OFe).

[0090] Comparative Example 1

[0091] In this comparative example 1, the protein linker CHO-PEG2k-CHO was replaced by "MAL-PEG 2k -MAL", and without adding NaOH, the other steps were the same as in Example 1 to prepare IRMH-MAL-OFe nanoparticles.

[0092] Comparative Example 2

[0093] In this comparative example 2, the mass ratio of albumin to ferritin is 3:1, and the other steps are the same as those in example 1.

[0094] Structural characterization of protein nanoparticles in Experimental Example 1, Example 1 and Comparative Example 2

[0095] 1. The morphology and size of the small-sized albumin nanoparticles, small-sized ferritin nanoparticles and large-sized pH-responsive protein nanoparticles with deformability and dual-targeting capabilities prepared in Example 1 were characterized by transmission electron microscopy (TEM).

[0096] like Figure 2 As shown in A, the synthesized small-sized albumin nanoparticles are uniformly spherical in shape and are approximately 10 nm in size. Figure 2 As shown in B, the synthesized small-sized ferritin nanoparticles also have a uniform spherical shape, good dispersion, and a size of about 10 nm.

[0097] like Figure 2As shown in C, the synthesized pH-responsive protein nanoparticles with deformability and dual-targeting capabilities are larger in size than single albumin nanoparticles and ferritin nanoparticles, and are approximately 50 nm in size.

[0098] 2. The TEM image of the pH-responsive and deformable dual-targeted nanoprotein finally prepared in Comparative Example 2 is as follows: Figure 7 As shown, it indicates that the size of the prepared nanoparticles is too large and the proportion of ferritin is too high.

[0099] In addition, when the mass ratio of albumin nanoparticles to ferritin nanoparticles is 3:1 or 4:1, and the other steps are the same as in Example 1, the morphology of the prepared pH-responsive deformable dual-targeted nanoprotein is substantially similar to that of Example 2C.

[0100] In summary, when the mass ratio of albumin nanoparticles to ferritin nanoparticles is in the range of (3:1)-(5:1), the size of the prepared nanoparticles is relatively uniform, 50-70 nm. If the mass ratio is too large or too small, the size of the nanoparticles may be too large.

[0101] pH responsiveness characterization of protein nanoparticles in Experimental Example 2, Example 1 and Comparative Example 1

[0102] Transmission electron microscopy (TEM) was used to characterize the CHO-PEG 2k -CHO-linked large-sized protein nanoparticles (Example 1 and Comparative Example 1) and Comparative Example 1 by MAL-PEG 2k -MAL-linked large-sized protein nanoparticles; CHO-PEG 2k -CHO connected to form large-sized protein nanoparticles, the imine bonds between proteins are pH-responsive, and the MAL-PEG 2k -MAL-linked large-sized protein nanoparticles form irreversible thioether bonds between proteins and are not pH-responsive.

[0103] like Figure 3 As shown:

[0104] When pH = 7.4, whether it is through CHO-PEG 2k -CHO or MAL-PEG 2k -MAL connection can form large-sized protein nanoparticles. When pH = 6.5, CHO-PEG 2k -CHO-linked large-sized protein nanoparticles gradually disintegrated and dispersed into multiple small units of protein nanoparticles. 2kThe large-sized protein nanoparticles formed by -MAL connection are still not affected by the slightly acidic environment and maintain a relatively stable large-sized protein nanoparticle structure.

[0105] Application Example 1: In vitro experiment

[0106] An in vitro co-culture experiment was carried out using CT26 tumor cells and the prepared pH-responsive deformable and dual-targeting protein nanoparticles (IRMH-CHO-OFe) to systematically study the anti-tumor properties of the nanoparticles (IRMH-CHO-OFe) in vitro.

[0107] Including: Tumor cytotoxicity. First, CT26 cells were seeded in 96-well plates (1×10 5 Cells / well) were cultured in complete 1640RPMI medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) for 24 hours. To verify that IRMH-CHO-OFe responds to the weakly acidic pH of the tumor microenvironment, the protein nanoparticles (IRMH-CHO-OFe) prepared in Example 1 with pH-responsive deformability and dual-targeting ability were co-cultured with CT2 tumor cells in medium with pH values ​​of 7.4 and 6.5 for 24 hours, and then 10% CCK8 solution was added and incubated for 1.5 hours, and the absorbance of each well at a wavelength of 450nm was measured using an enzyme reader.

[0108] like Figure 4 As shown in A, under the condition of pH = 7.4, the toxicity of protein nanoparticles IRMH-CHO-OFe at different concentrations to CT26 tumor cells is very small. Only under extremely high concentration conditions can it approach the half-lethal toxicity to CT26 tumor cells. Figure 4 As shown in Figure B, under the same concentration conditions, at pH = 6.5, the toxic effect of protein nanoparticles IRMH-CHO-OFe on CT26 tumor cells was significantly enhanced. This is because IRMH-CHO-Ofe has pH-responsive deformability and decomposes into small-sized units in a slightly acidic environment, which enhances the targeting and internalization of ferritin loaded with the chemotherapy drug oxaliplatin. At the same time.

[0109] like Figure 4 This is further confirmed by the fact that the MAL-PEG prepared in Comparative Example 1 has no pH responsiveness. 2k -MAL-connected large-sized protein nanoparticles (IRMH-MAL-OFe) are less toxic than the pH-responsive CHO-PEG prepared in Example 1 2k-CHO connected to form large-sized protein nanoparticles (IRMH-CHO-OFe). However, the toxicity of the small-sized ferritin nanoparticles loaded with oxaliplatin Oxa@Fep prepared by step 3 in Example 1 is similar to that of the large-sized protein nanoparticles (IRMH-CHO-OFe) prepared in Example 1 of the present invention, indicating that large proteins can be dissociated into small proteins in the tumor microenvironment, and have the same killing ability as small-sized ferritin nanoparticles.

[0110] Application Example 2: Characterization of the in vitro anti-tumor effect of pH-responsive protein nanoparticles with deformability and dual-targeting capabilities

[0111] In vitro co-culture experiments were carried out using CT26 tumor cells and the prepared pH-responsive deformable and dual-targeting protein nanoparticles (IRMH-CHO-OFe). The supernatant obtained from the co-culture was further co-cultured with immune cell macrophages in vitro to systematically study the anti-tumor effect of the nanoparticles (IRMH-CHO-OFe) in vitro.

[0112] Including: comet assay and immunofluorescence assay to evaluate the degree of DNA damage in tumor cells. First, CT26 cells were seeded in 6-well plates (2×10 5 The cells were cultured in complete 1640RPMI medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) for 24 hours. To verify the degree of DNA damage of tumor cells after incubation of IRMH-CHO-OFe with tumor cells, the supernatant was removed and the cells were collected after 24 hours of co-culture of IRMH-CHO-OFe and CT26 cells, and the DNA damage of CT26 tumor cells was determined by comet assay and immunofluorescence.

[0113] The results are as follows Figure 5 As shown in A, after treatment with IRMH-CHO-OFe protein nanoparticles, the comet assay results of CT26 tumor cells showed significant tailing compared with the blank control group, confirming its damage to tumor cell DNA. The quantitative results are shown in Figure 5 As shown in C.

[0114] In addition, immunofluorescence staining further verified the damage of IRMH-CHO-OFe protein nanoparticles to tumor cell DNA, and γ-H2AX was used as a marker of DNA double-strand breaks, such as Figure 5 As shown in B, the signal of γ-H2AX in CT26 tumor cells treated with IRMH-CHO-OFe was enhanced, which once again confirmed its DNA damage to tumor cells and its effective killing function on tumor cells.

[0115] ELISA was used to determine the content of cytokines TNF-α, IL-6, and IFN-β secreted by macrophages. First, CT26 and macrophages were seeded in 6-well plates (2×105 cells / well) and cultured in complete 1640RPMI medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) for 24 hours. Then, IRMH-CHO-OFe was co-cultured with CT26 tumor cells for 24 hours, and the cell supernatant obtained by co-culture was co-cultured with macrophages for 24 hours. The macrophage supernatant obtained after co-culture was collected, and the content of cytokines secreted by macrophages was determined by ELISA.

[0116] like Figure 5 As shown in D, compared with IRMH-MAL-OFe, which is not pH-responsive, the content of TNF-α secreted by macrophages was significantly increased after treatment with IRMH-CHO-OFe, verifying that after the IRMH-CHO-OFe protein nanoparticles dissociated into small protein nanoparticles after pH response, they promoted the endocytosis of albumin nanoparticles loaded with IRG1-IN-1 by macrophages. The downregulation of the IRG1 gene promoted the polarization of macrophages into pro-inflammatory M1 macrophages, further promoting the increased secretion of inflammatory factors.

[0117] like Figure 5 As shown in Figure E, a significant increase in the secretion of IL-6 by macrophages was observed after IRMH-CHO-OFe treatment. In addition, the secretion of itaconic acid caused by IRG1 can inhibit the activation of STING, and after IRMH-CHO-OFe treatment, the IRG1 gene of macrophages was downregulated, thereby reducing the secretion of itaconic acid and further promoting the activation of STING. Figure 5 As shown in F, after IRMH-CHO-OFe treatment, the content of IFN-β, a downstream cytokine of the STING activation pathway in macrophages, was significantly increased.

[0118] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0119] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0120] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a pH-responsive deformable dual-targeting nanoprotein, characterized in that: The method comprises: After the carboxylated mannose was activated by the EDC / NHS system, it was reacted with the albumin solution and purified by dialysis to obtain the mannose-modified albumin. The mannose-modified albumin is mixed with the inhibitor IRG1-IN-1, and the IRG1-IN-1 is loaded by hydrophobic interaction to prepare mannose albumin nanoparticles; Ferritin nanoparticles were prepared by using ferritin as a carrier and loading oxaliplatin via solvent replacement method. Using CHO-PEG2k-CHO as a linker, the mannose albumin nanoparticles and the ferritin nanoparticles are cross-linked through a Schiff base reaction to form a large-sized pH-responsive dual-targeting nanoprotein.

2. The preparation method according to claim 1, characterized in that: The specific conditions of the EDC / NHS activation system are: The molar ratio of EDC, NHS and carboxylated mannose is (1.5-2.5): (1.5-2.5): 1, the activation reaction time is 6-10 hours, and the reaction temperature is 20-25°C.

3. The preparation method according to claim 2, characterized in that: The concentration of the carboxylated mannose is 0.5-2 mM, and the mass ratio of albumin to carboxylated mannose is 1:(0.1-0.5).

4. The preparation method according to claim 1, characterized in that: The method for loading IRG1-IN-1 comprises: dissolving IRG1-IN-1 in ethanol, adding dropwise to a mannose albumin solution containing β-mercaptoethanol at a rate of 5-10 μL / s, stirring for reaction for 4-8 hours, and then centrifuging for purification.

5. The preparation method according to claim 1, characterized in that: The conditions for loading oxaliplatin are: The concentration of ferritin is 1-3 mg / mL, the molar ratio of oxaliplatin to ferritin is (5-20):1, and the solvent is a mixed system of DMF and ultrapure water, wherein the volume ratio of DMF to ultrapure water is 1:10-1:

20.

6. The preparation method according to claim 1, characterized in that: The conditions of the Schiff base reaction include: The reaction pH is 8-10, the mass ratio of albumin nanoparticles to ferritin nanoparticles is (3:1)-(5:1), and the reaction time is 4-8 hours.

7. The preparation method according to claim 6, characterized in that: The added amount of the CHO-PEG2k-CHO is 5%-15% of the total mass of albumin and ferritin.

8. A pH-responsive deformable dual-targeting nanoprotein prepared by any method of claims 1-7, characterized in that: (a) Maintaining large-size structures of 40-60 nm at pH ≥ 7.4; (b) dissociated into 10-15 nm mannose albumin nanoparticles and ferritin nanoparticles at pH ≤ 6.5; (c) Mannosalbumin nanoparticles target macrophages and inhibit the IRG1-ITA pathway, while ferritin nanoparticles target tumor cells and induce DNA damage.

9. The dual-targeting nanoprotein according to claim 8, characterized in that: The mannose modification degree of the mannose albumin nanoparticles is that 3-8 mannose molecules are connected to each albumin molecule; The oxaliplatin loading amount of the ferritin nanoparticles is 8%-15% (w / w), and the encapsulation rate of IRG1-IN-1 in the mannose albumin nanoparticles is ≥85%.

10. Use of the dual-targeting nanoprotein according to any one of claims 8 to 9 in the preparation of anti-tumor drugs, characterized in that: (i) Tumor microenvironment-specific dual targeting via pH-responsive dissociation; (ii) synchronously kill tumor cells and regulate macrophage metabolism to relieve immunosuppression; (iii) Activate the STING pathway and promote the secretion of inflammatory factors TNF-α, IL-6, and IFN-β.

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