A protein nanoparticle with deformability and dual targeting ability in response to pH and a preparation method and application thereof

Abstract

The application discloses a pH-responsive deformable double-targeting nano-protein and a preparation method and application thereof, and the method comprises the following steps: after carboxylated mannose is activated through an EDC / NHS system, the carboxylated mannose is reacted with an albumin solution, and mannose-modified albumin is obtained through dialysis purification; the mannose-modified albumin is mixed with an inhibitor IRG1-IN-1, and IRG1-IN-1 is loaded through hydrophobic interaction to obtain mannose albumin nanoparticles; ferritin is used as a carrier, and oxaliplatin is loaded through a solvent displacement method to obtain ferritin nanoparticles; CHO-PEG2k-CHO is used as a connecting agent, and the mannose albumin nanoparticles and the ferritin nanoparticles are crosslinked through a Schiff base reaction to form large-size pH-responsive double-targeting nano-proteins. The double-targeting separable characteristics of the nano-proteins can realize specific treatment, kill tumor cells, interfere with immune cell metabolism, block metabolic immunosuppression, and promote anti-tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a protein nanoparticle with deformability and dual-targeting capability in response to pH, its preparation method and application. Background Technology

[0002] With the continuous development of cancer treatment strategies, traditional treatments such as surgery, radiotherapy, and chemotherapy still occupy a mainstream position. However, these methods often overlook 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, and metabolically demanding tumor microenvironment significantly impacts the function of immune cells. In particular, metabolic alterations in important immune cells such as macrophages and T cells within the tumor microenvironment suppress their normal immune responses, leading to ineffective anti-tumor immunity and poor prognosis. For instance, tumor cell metabolism alters macrophage metabolic pathways, promoting the transformation of macrophages into an immunosuppressive phenotype and inhibiting their ability to recognize and clear tumors. Furthermore, the activity of immunosuppressive cells such as tumor-associated macrophages and regulatory T cells also enhances tumor immune escape capabilities through metabolic changes. These metabolic disorders of immune cells are closely related to tumor growth and metastasis, directly contributing to unsatisfactory treatment outcomes and poor patient prognosis.

[0004] Therefore, it is necessary and urgent to develop a multifunctional dual-targeting 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 this invention is to provide a pH-responsive, deformable, and dual-targeting protein nanoparticle, its preparation method, and its application. The pH-responsive deformability makes it highly specific. After actively targeting the tumor microenvironment and deforming, it can target macrophages and tumor cells separately, improving the efficiency of treatment. It compensates for the immunosuppressive microenvironment caused by targeting the tumor alone, thereby comprehensively improving the efficiency of treatment and realizing a clever integration of chemotherapy and immunotherapy.

[0006] In a first aspect of the present invention, a protein nanoparticle with deformability and dual-targeting capability in response to pH and a method for preparing the same are provided, the method comprising:

[0007] Carboxylated mannose was activated using an EDC / NHS system, reacted with an albumin solution, and purified by dialysis to obtain mannose-modified albumin.

[0008] The mannose-modified albumin was mixed with the inhibitor IRG1-IN-1, and IRG1-IN-1 was loaded through hydrophobic interaction to prepare mannose albumin nanoparticles.

[0009] Ferritin nanoparticles were prepared by loading oxaliplatin onto ferritin as a carrier via a solvent displacement method.

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

[0011] Furthermore, the specific conditions for the EDC / NHS activation system are as follows:

[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℃.

[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] Furthermore, the method for loading IRG1-IN-1 includes:

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

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

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

[0018] Furthermore, the conditions for the Schiff base reaction include:

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

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

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

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

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

[0024] (c) Mannoalbumin 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 such that each albumin molecule is linked to 3-8 mannose molecules;

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

[0027] In a third aspect of the invention, the application of the dual-targeting nanoprotein in the preparation of antitumor drugs is provided.

[0028] (i) Achieving specific dual targeting of the tumor microenvironment through pH-responsive dissociation;

[0029] (ii) Simultaneously kills tumor cells and regulates 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 protein nanoparticle with pH-responsive deformability and dual targeting capability, which has the ability to actively target tumors and can deform from a large size to a small size in response to the microacidic environment of tumors, targeting tumor cells and macrophages respectively, thus exhibiting strong specificity for precise cell regulation.

[0033] 2. This invention provides a pH-responsive protein nanoparticle with deformability and dual-targeting capabilities. During tumor treatment, it initiates a series of cascade reactions in response to the acidic pH of the tumor microenvironment, including:

[0034] Through CHO-PEG 2kLarge protein nanoparticles formed by -CHO linkages reach the acidic environment of the tumor, where Schiff bases break, releasing smaller protein nanoparticles targeting tumors and macrophages, respectively. Ferritin nanoparticles, with transferrin receptors, actively target tumor cells and are internalized by them, releasing the chemotherapy drug oxaliplatin, promoting tumor cell DNA damage, tumor cell necrosis, and the release of mtDNA. Meanwhile, albumin nanoparticles containing mannose actively target macrophages with mannose receptors and are internalized, releasing the IRG1-IN-1 drug, interfering with the IRG1-ITA pathway, downregulating IRG1 expression, and thus reducing the production of the immunosuppressive metabolite itaconic acid (ITA) by macrophages. The mtDNA released by tumor cell death promotes the activation of STING in macrophages, and the reduction in itaconic acid levels further alleviates the inhibition of the STING pathway, further promoting STING activation and thus promoting anti-tumor immunity.

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

[0036] 4. The present invention provides a protein nanoparticle with deformability and dual-targeting capability in response to pH and a method for preparing the same. By deforming in response to pH, it promotes precise dual-targeting, avoiding the limitations of single tumor cell regulation or single immune cell regulation, and avoiding the defects of insufficient targeting when dual-targeting proteins are simultaneously introduced.

[0037] 5. The present invention provides a protein nanoparticle with deformability and dual targeting capability in response to pH and a method for preparing the same. The preparation is simple and convenient, and the obtained protein nanoparticles are stable and highly efficient and precise in tumor treatment. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

[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-targeting protein nanoparticles (IRMH-CHO-OFe); among them, Figure 2 A is a TEM image of small 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 obtained through CHO-PEG. 2k TEM image of large protein nanoparticles formed by -CHO linkages;

[0041] Figure 3 For different pH conditions, CHO-PEG 2k Large protein nanoparticles formed by -CHO linkage and through MAL-PEG 2k TEM image of large protein nanoparticles formed by MAL linkages;

[0042] Figure 4 In vitro tumor toxicity characterization of protein nanoparticles with deformability and dual targeting capabilities in response to pH; among which, Figure 4 A represents the toxicity of different concentrations of pH-responsive, deformable, and dual-targeting protein nanoparticles to CT26 tumor cells at a pH of 7.4. Figure 4 B represents the toxicity of different concentrations of pH-responsive, deformable, and dual-targeting protein nanoparticles to CT26 tumor cells at a pH of 6.5. Figure 4 C represents the toxicity results of different groups of materials to CT26 tumor cells. The groups are the blank control group, small albumin nanoparticles (IRG1-IN-1@MHSA), small ferritin nanoparticles (Oxa@Fep), and those processed by MAL-PEG. 2k Large protein nanoparticles formed by MAL linkage (IRMH-MAL-OFe) and via CHO-PEG 2k Large protein nanoparticles formed by -CHO linkage (IRMH-CHO-OFe);

[0043] Figure 5 Characterization of the tumor cell killing effect of protein nanoparticles with deformability and dual targeting capabilities in response to pH; among which, Figure 5 A represents the comet assay used to assess the DNA damage to CT26 tumor cells caused by different materials after incubation. Figure 5B represents the degree of DNA damage induced by different materials on CT26 tumor cells after incubation with them, as determined by immunofluorescence. Figure 5 C represents the quantitative assessment of DNA damage in CT26 tumor cells caused by different materials. Figure 5 D represents the incubation of different materials with CT26 followed by incubation with macrophages, and the determination of the content of the inflammatory factor TNF-α produced in the macrophage supernatant. Figure 5 E represents the incubation of different materials with CT26 followed by incubation with macrophages, and the determination of the content of the inflammatory factor IL-6 produced in the macrophage supernatant. Figure 5 F represents the supernatant of different materials incubated with CT26, which was then incubated with macrophages to determine the content of IFN-β produced in the macrophage supernatant;

[0044] Figure 6 This is a flowchart illustrating a method for preparing protein nanoparticles with deformability and dual targeting capabilities in response to pH, as provided in an embodiment of the present invention.

[0045] Figure 7 This is a TEM image of the nanoparticles prepared in Comparative Example 2. Detailed Implementation

[0046] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0047] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0048] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

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

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

[0051] CHO-PEG 2k-CHO, Name: Aldehyde-based polyethylene glycol, Aldehyde-based PEG, Alias: Dialdehyde-based polyethylene glycol, English Name: CHO-PEG-CHO ALD-PEG-ALD, Molecular weight (PEG) is 2000, purchased from Chongqing Yusi Pharmaceutical.

[0052] This invention provides a protein nanoparticle that exhibits deformability and dual-targeting capability in response to pH, such as... Figure 6 As shown, the overall approach is as follows:

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

[0054] S1. After activating carboxylated mannose, it is modified onto albumin, and after dialysis and freeze-drying, mannose-modified albumin is obtained.

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

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

[0057] Equivalent molar ratios of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were completely dissolved in ultrapure aqueous solution. Then, carboxymannose aqueous solution was added, and the mixture was stirred at room temperature for 8 hours to obtain carboxy-activated mannose. Further, the activated carboxy-modified mannose was modified onto albumin. A certain amount of albumin was dissolved in ultrapure water and ultrasonically dispersed. The albumin solution was then added to the carboxymannose solution with activated carboxyl groups, stirred, dialyzed for 2 days, and lyophilized to obtain mannose-modified albumin.

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

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

[0060] The stirring time for adding albumin solution to carboxymannose solution with activated carboxyl groups is 24-48 hours.

[0061] S2. Dissolve mannose-modified albumin in water, add the inhibitor IRG1-IN-1 and stir to obtain small-sized mannose-modified albumin nanoparticles.

[0062] 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 and ultrasonically dispersed. 35 μl of β-mercaptoethanol was added and stirred for 2 min. IRG1-IN-1 dissolved in ethanol was slowly added dropwise to the albumin solution. After stirring for 6 h, centrifugation was performed to obtain small-sized albumin nanoparticles loaded with IRG1-IN-1.

[0064] The solvent for the IRG1-IN-1 solution is ethanol, and the dropping rate is controlled at 5-10 μL / s. Nanoparticles formed at rates within this range are relatively uniform. If the dropping rate is less than 5 μL / s or greater than 10 μL / s, the formed nanoparticles are prone to irregularity, which is not conducive to subsequent protein-protein linkage reactions.

[0065] S3. Using ferritin as a carrier, the chemotherapy drug oxaliplatin was loaded to obtain small-sized ferritin nanoparticles.

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

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

[0068] A certain amount of oxaliplatin was dissolved in DMF and ultrasonically dispersed. Ferritin (2 mg / mL, 500 μL) was added to 10 mL of ultrapure water and ultrasonically dispersed. Then, oxaliplatin dissolved in DMF was added. After stirring at room temperature for 12 h, small-sized ferritin nanoparticles loaded with oxaliplatin were obtained.

[0069] S4. Add CHO-PEG 2k -CHO was used as a linker to link small albumin nanoparticles and ferritin nanoparticles through a Schiff base reaction, resulting in large-sized pH-responsive dual-targeting nanoproteins.

[0070] The conditions for 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 small-sized albumin to 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] Step S4 specifically includes:

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

[0075] The reason for adding 5-15 μL (preferably 10 μL) of 1M NaOH is to adjust the pH of the solution, CHO-PEG 2k The Schiff base reaction occurs when two proteins are linked by -CHO. The Schiff base reaction is more stable under alkaline conditions (pH 8-10), making it easier for the two proteins to be linked into large-sized pH-responsive dual-targeting protein nanoparticles.

[0076] According to another typical embodiment of the present invention, protein nanoparticles with deformability and dual targeting capabilities in response to pH are provided, prepared by the method described above.

[0077] According to another typical embodiment of the present invention, the application of the aforementioned pH-responsive, deformable, and dual-targeting protein nanoparticles as an antitumor drug is provided.

[0078] This pH-responsive, deformable, and dual-targeting protein nanoparticle can actively cleave into two functionally independent subunits in the acidic environment of a tumor, targeting tumor cells and macrophages respectively. This "environment-triggered functional subtyping" mechanism overcomes the limitations of traditional single- or dual-targeting carriers that rely solely on a single response to release the drug. Furthermore, the deformability mechanism and dual targeting synergistically enhance each other, enabling the killing of tumor cells while precisely regulating macrophage metabolism, downregulating the expression of the macrophage IRG1 gene, thereby downregulating the production of the immunosuppressive metabolite itaconic acid, de-inhibiting the inhibitory effect of itaconic acid on other immune cells, and promoting anti-tumor immunity. In addition, the nanoparticle cleavage via pH-sensitive imine bonds responds to the acidic tumor environment, and the deformed, smaller subunits more easily penetrate the dense tumor matrix, further increasing its penetration depth and solving the problem of traditional nanoparticles being limited by the EPR effect. Moreover, the natural protein carrier has low immunogenicity, is more easily cleared from the body, reduces the risk of long-term toxicity, and has strong biocompatibility. Figure 1 As shown, the design and development of such pH-responsive, deformable, and dual-targeting protein nanoparticles provides a new strategy for the efficient treatment of tumors and offers a new approach for the ingenious combination of chemotherapy and immunotherapy.

[0079] The following will provide a detailed description of a protein nanoparticle with deformability and dual targeting capabilities in response to pH, based on embodiments and experimental data.

[0080] Example 1: pH-responsive deformable dual-targeting nanoprotein and its preparation method

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

[0082] Equivalent molar ratios of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were completely dissolved in ultrapure aqueous solution, with the molar ratio of EDC, NHS, and carboxymannose being 2:2:1. Then, an aqueous solution of carboxymannose was added, and the mixture was stirred at room temperature for 8 hours to obtain a carboxy-activated mannose solution. Further, 10 mg of albumin was dissolved in ultrapure water and ultrasonically dispersed (the mass-to-volume ratio of albumin to ultrapure water was 1:1). The albumin solution was then added to the carboxy-activated mannose solution (the volume ratio of albumin solution to the carboxy-activated mannose solution was 2:1), where the concentration of the hydroxymannose aqueous solution could be 0.5 mM. After stirring for 24 hours, the mixture was dialyzed for 2 days and then lyophilized to obtain mannose-modified albumin.

[0083] 1 mg of IRG1-IN-1 was dissolved in 500 μL of ethanol and ultrasonically dispersed. 5 mg of mannose-modified albumin was dissolved in 5 mL of ultrapure water and ultrasonically dispersed. 35 μL of β-mercaptoethanol was added and stirred for 2 min. Then, IRG1-IN-1 dissolved in ethanol was slowly added dropwise to the albumin solution. After stirring for 6 h, centrifugation was performed to obtain small-sized albumin nanoparticles loaded with IRG1-IN-1.

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

[0085] 1 mg of IRG1-IN-1 (the inhibitor IRG1-IN-1 of this invention was purchased from MCE, catalog number HY-148335) was dissolved in 500 μL of ethanol and ultrasonically dispersed. 5 mg of mannose-modified albumin was dissolved in 5 mL of ultrapure water and ultrasonically dispersed. 35 μL of β-mercaptoethanol was added and stirred for 2 min. The IRG1-IN-1 dissolved in ethanol was then slowly added dropwise to the albumin solution (dropping rate 5-10 μL / s). After stirring for 6 h, centrifugation was performed to obtain small-sized albumin nanoparticles loaded with IRG1-IN-1.

[0086] 3. Small-sized oxaliplatin-loaded ferritin nanoparticles and their preparation methods

[0087] 1 mg of oxaliplatin was dissolved in 500 μL of DMF and ultrasonically dispersed to obtain a DMF solution containing oxaliplatin. Ferritin (2 mg / mL, 500 μL) 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 their preparation methods

[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. Then, 10 μL of NaOH (1 M) was added to adjust the pH to 8.2. After stirring for 2 min, ethanol solution was added and stirred for 3 h. Then, 200 μL (100 mM) of protein linker CHO-PEG2k-CHO was added. After reacting for 6 h, the mixture was centrifuged and washed with a 10 W ultrafiltration tube to obtain the pH-responsive deformable dual-targeting nanoprotein (IRMH-CHO-OFe).

[0090] Comparative Example 1

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

[0092] Comparative Example 2

[0093] In Comparative Example 2, the mass ratio of albumin to ferritin was 3:1, and the other steps were the same as 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 albumin nanoparticles, small ferritin nanoparticles and large protein nanoparticles with deformability and dual targeting ability in response to pH were characterized by transmission electron microscopy (TEM) in Example 1.

[0096] like Figure 2 As shown in Figure A, the synthesized small-sized albumin nanoparticles exhibit a uniform spherical shape with a size of approximately 10 nm; Figure 2 As shown in B, the synthesized small-sized ferritin nanoparticles also exhibit a uniform spherical shape, good dispersibility, and a size of approximately 10 nm.

[0097] like Figure 2As shown in C, the synthesized protein nanoparticles, which are deformable and have dual targeting capabilities in response to pH, are larger in size than single albumin nanoparticles and ferritin nanoparticles, with a size of approximately 50 nm.

[0098] 2. TEM image of the pH-responsive deformable dual-targeting nanoprotein finally prepared in Comparative Example 2 is shown below. Figure 7 As shown, the nanoparticles obtained are too large in size and contain too much ferritin.

[0099] Furthermore, when the mass ratio of albumin nanoparticles to ferritin nanoparticles is 3:1 or 4:1, and other steps are the same as in Example 1, the morphology of the pH-responsive deformable dual-targeting nanoprotein prepared is basically 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) to (5:1), the resulting nanoparticles have a relatively uniform size of 50-70 nm. If the mass ratio is too large or too small, it is easy to cause the nanoparticles to be too large.

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

[0102] Characterization of CHO-PEG by transmission electron microscopy (TEM) 2k Large protein nanoparticles formed by -CHO linkage (Example 1 and Comparative Example 1) and the MAL-PEG-linked nanoparticles in Comparative Example 1 2k The morphology of large protein nanoparticles formed by -MAL linkage; via CHO-PEG 2k Large protein nanoparticles formed by -CHO linkages exhibit pH-responsive imine bonds between proteins, while MAL-PEG... 2k Large protein nanoparticles formed by MAL linkages have irreversible thioether bonds between proteins and do not exhibit pH responsiveness.

[0103] like Figure 3 As shown:

[0104] At pH = 7.4, regardless of whether it is via CHO-PEG 2k -CHO or MAL-PEG 2k -MAL linkages can all form large-sized protein nanoparticles. And at pH 6.5, CHO-PEG... 2k The large protein nanoparticles formed by -CHO linkages gradually disintegrate and disperse into multiple smaller protein nanoparticle units. However, through MAL-PEG... 2kThe large-sized protein nanoparticles formed by MAL linkages remain unaffected by the slightly acidic environment, maintaining a relatively stable structure.

[0105] Application Example 1: In vitro experiments

[0106] The in vitro antitumor properties of CT26 tumor cells (IRMH-CHO-OFe), a protein nanoparticle with pH-responsive deformability and dual-targeting ability, were systematically studied by co-culturing them in vitro.

[0107] Including: tumor cytotoxicity. First, CT26 cells were seeded in 96-well plates (1×10⁻⁶ cells / well). 5 Cells / well were cultured and incubated in complete 1640 RPMI medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) for 24 h. To verify the response of IRMH-CHO-OFe to the weakly acidic pH of the tumor microenvironment, CT2 tumor cells with pH-responsive deformability and dual-targeting ability (IRMH-CHO-OFe) prepared in Example 1 were co-cultured in mediums with pH 7.4 and 6.5 for 24 h, and then 10% CCK8 solution was added and incubated for 1.5 h. The absorbance of each well at 450 nm was measured using a microplate reader.

[0108] like Figure 4 As shown in Figure A, under pH 7.4 conditions, different concentrations of the protein nanoparticle IRMH-CHO-OFe exhibited very low toxicity to CT26 tumor cells. Only at extremely high concentrations did it approach the LD50 toxicity level for CT26 tumor cells. However, as... Figure 4 As shown in Figure B, under the same concentration conditions and at pH 6.5, the cytotoxic effect of the protein nanoparticles IRMH-CHO-OFe on CT26 tumor cells was significantly enhanced. This is because IRMH-CHO-OFe exhibits pH-responsive deformability, decomposing into smaller units in a slightly acidic environment, thus enhancing the targeting and internalization of ferritin loaded with the chemotherapeutic drug oxaliplatin. Simultaneously...

[0109] like Figure 4 Figure C further confirms this, showing that the MAL-PEG prepared in Comparative Example 1 lacked pH responsiveness. 2k The MAL-linked large-size protein nanoparticles (IRMH-MAL-OFe) are less toxic than the pH-responsive CHO-PEG prepared in Example 1. 2kLarge-sized protein nanoparticles (IRMH-CHO-OFe) were formed by linking -CHO. However, the toxicity of the small-sized ferritin nanoparticles Oxa@Fep loaded with oxaliplatin prepared by step 3 in Example 1 was similar to that of the large-sized protein nanoparticles (IRMH-CHO-OFe) prepared in Example 1 of this invention, indicating that large proteins can dissociate into small proteins in the tumor microenvironment, possessing the same killing ability as small-sized ferritin nanoparticles.

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

[0111] We conducted an in vitro co-culture experiment using CT26 tumor cells and prepared pH-responsive, deformable, and dual-targeting protein nanoparticles (IRMH-CHO-OFe). The supernatant obtained from the co-culture was then further co-cultured with immune cells (macrophages) to systematically study the anti-tumor effect of the nanoparticles (IRMH-CHO-OFe) in vitro.

[0112] This includes comet assays and immunofluorescence assays to assess the degree of DNA damage in tumor cells. First, CT26 cells were seeded in 6-well plates (2 × 10⁻⁶). 5 Cells / well were cultured in complete 1640 RPMI medium (containing 10% fetal bovine serum and 1% penicillin and streptomycin) for 24 h. To verify the degree of DNA damage to tumor cells after incubation with IRMH-CHO-OFe, CT26 cells were co-cultured with IRMH-CHO-OFe for 24 h, the supernatant was removed, and the cells were collected for DNA damage determination using comet assay and immunofluorescence.

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

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

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

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

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

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

[0119] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0120] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends 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: The carboxylated mannose is activated by an EDC / NHS system and then reacted with an albumin solution, and the mannose-modified albumin is obtained by dialysis purification; The mannose-modified albumin is mixed with an inhibitor IRG1-IN-1, and the IRG1-IN-1 is loaded by hydrophobic interaction to prepare mannose-albumin nanoparticles; and the ferritin is used as a carrier to load oxaliplatin by a solvent displacement method to prepare ferritin nanoparticles; The mannose-albumin nanoparticles and the ferritin nanoparticles are crosslinked by Schiff base reaction to form large-size pH-responsive double-targeted nano-proteins by using CHO-PEG2k-CHO as a linker. The mass ratio of the albumin nanoparticles to the ferritin nanoparticles is 5:

1.

2. The production method according to claim 1, characterized by, The specific conditions of the EDC / NHS activation system are as follows: the molar ratio of EDC, NHS to 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℃.

3. The production method according to claim 2, characterized by, 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 method of claim 1, wherein, The method for loading IRG1-IN-1 comprises the following steps: dissolving IRG1-IN-1 in ethanol, adding the IRG1-IN-1 dropwise into a mannose-albumin solution containing β-mercaptoethanol at a rate of 5-10 μL / s, stirring for 4-8 hours, and then centrifuging and purifying.

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

20.

6. The method of claim 1, wherein, The conditions of the Schiff base reaction include: the reaction pH is 8-10, and the reaction time is 4-8 hours.

7. The production method according to claim 6, wherein The addition amount of the CHO-PEG2k-CHO is 5%-15% of the total mass of the albumin and the ferritin.

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

9. The dual-targeting nanoprotem of claim 8, wherein The mannose modification degree of the mannose-albumin nanoparticles is 3-8 mannose molecules per albumin molecule. The oxaliplatin loading amount of the ferritin nanoparticles is 8%-15% (w / w), and the encapsulation efficiency of IRG1-IN-1 in the mannose-albumin nanoparticles is ≥85%.

10. The use of the dual-targeting nanoprotein according to any one of claims 8-9 in the preparation of an anti-CT26 tumor drug, characterized in that, (i) achieving tumor microenvironment-specific double targeting by pH-responsive dissociation; (ii) synchronously killing tumor cells and regulating macrophage metabolism to relieve immunosuppression; (iii) activating the STING pathway and promoting the secretion of inflammatory factors TNF-α, IL-6 and IFN-β.

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