A complex supramolecular gel composition based on ascorbic acid and gels, methods of preparation and uses thereof

Abstract

The present application relates to the field of pharmaceutical preparations and drug delivery, in particular to a complex ascorbic acid-based supramolecular gel composition and gels, preparation method and application thereof. The complex ascorbic acid-based supramolecular gel composition of the present application comprises an ascorbic acid derivative and a polymer nanoparticle; the ascorbic acid derivative is obtained by esterification of ascorbic acid and C 10 ~C 20 alkanoic acid; the composition of the shell of the polymer nanoparticle comprises polydopamine, and the composition of the core comprises hemoglobin and an iron death inducer. The complex ascorbic acid-based supramolecular gel of the present application has the advantages of good in-vivo stability and good anti-tumor effect.

Description

Technical Field

[0001] This application relates to the fields of pharmaceutical formulation and drug delivery, and particularly to a composite supramolecular gel composition based on ascorbic acid, the gel, preparation method and application thereof, as well as an antitumor drug formulation and its preparation method. Background Technology

[0002] Ascorbic acid (AA), also known as vitamin C, is an essential micronutrient for the human body. It possesses various biological functions, including antioxidant activity, promotion of collagen synthesis, immune enhancement, aiding iron absorption, and participation in neurotransmitter synthesis. Current research indicates that in mouse tumor models of breast cancer, colorectal cancer, pancreatic cancer, and melanoma, intraperitoneal injection of high doses of ascorbic acid significantly inhibits tumor growth, suggesting that ascorbic acid also has potential anti-cancer effects. The anti-cancer mechanism of ascorbic acid mainly involves its oxidized form—dehydroascorbic acid (DHA)—inducing cellular oxidative stress and triggering ferroptosis.

[0003] However, there are still many limitations to using ascorbic acid as an anticancer drug in clinical practice: First, the high water solubility of ascorbic acid leads to its rapid metabolism in the body, making it difficult to achieve high concentrations of accumulation in tumor tissues; second, the hypoxic microenvironment of tumor tissues is not conducive to the conversion of AA to DHA; these limitations seriously affect the antitumor efficacy of ascorbic acid.

[0004] Given the heterogeneity of tumor cells and the complexity of the tumor microenvironment, anti-tumor therapy may need to act on multiple targets and pathways simultaneously to more comprehensively inhibit tumors, thereby improving treatment effectiveness and prolonging patient survival.

[0005] Therefore, it is necessary to develop an ascorbic acid drug formulation with good anti-tumor effects that can be used in combination with various anti-tumor active ingredients. Summary of the Invention

[0006] Based on this, one or more embodiments of this application provide an ascorbic acid-based composite supramolecular gel composition, its gel, preparation method, and application. The ascorbic acid-based composite supramolecular gel of this application has the advantages of good in vivo stability and good anti-tumor effect.

[0007] The technical solution of this application includes the following:

[0008] In a first aspect, the technical solution of the embodiments of this application provides a composite supramolecular gel composition based on ascorbic acid, including ascorbic acid derivatives and polymer nanoparticles;

[0009] The ascorbic acid derivative is obtained through ascorbic acid and C. 10 ~C 20 Alkyl acids are obtained through esterification.

[0010] The polymer nanoparticles have a core and a shell covering the core, the shell comprising polydopamine and the core comprising hemoglobin and ferroptosis inducer.

[0011] In the ascorbic acid-based composite supramolecular gel composition of this application embodiment, the ascorbic acid derivative is composed of ascorbic acid and C 10 ~C 20 Alkyl acids are obtained through esterification and contain ester bonds. The outer shell of the polymer nanoparticles is polydopamine, which allows the polymer nanoparticles to enter the cell through dopamine receptors. The core contains ferroptosis inducers and hemoglobin. Ferrroptosis inducers can promote cell death, and hemoglobin provides an oxygen environment after oxygenation. Since tumor tissue contains abundant esterases, the ascorbic acid-based composite supramolecular gel composition releases ascorbic acid and polymer nanoparticles under the action of esterases. Hemoglobin in the polymer nanoparticles compensates for the hypoxia of the tumor microenvironment by releasing oxygen, promoting the oxidation of ascorbic acid to dehydroascorbic acid. Subsequently, dehydroascorbic acid enters the cell and triggers ferroptosis by inducing cellular oxidative stress. Since the tumor cell interior is usually acidic, when the polymer nanoparticles enter the tumor cell, the polydopamine shell of the polymer nanoparticles undergoes protonation and structural relaxation in the acidic environment, thereby releasing ferroptosis inducers. Ferrroptosis inducers and dehydroascorbic acid synergistically exacerbate ferroptosis in tumor cells.

[0012] Further, the weight ratio of the ascorbic acid derivative to the polymer nanoparticles is 1:(0.03~0.05); and / or,

[0013] The ascorbic acid derivatives include at least one of palmitic ascorbate, capric ascorbate, lauric ascorbate, myristic ascorbate, and oleic ascorbate.

[0014] Furthermore, the ascorbic acid-based composite supramolecular gel composition satisfies at least one of the following conditions:

[0015] (1) The particle size of the polymer nanoparticles is 100 nm to 140 nm;

[0016] (2) The thickness of the outer shell is 5% to 10% of the particle size of the polymer nanoparticles;

[0017] (3) In the polymer nanoparticles, the mass percentage of the core is ≥80%.

[0018] Secondly, the technical solution of this application provides a composite supramolecular gel based on ascorbic acid, which is formed by the self-assembly of the composite supramolecular gel composition based on ascorbic acid described above in a solution environment.

[0019] The ascorbic acid-based composite supramolecular gel of this application embodiment utilizes the aforementioned ascorbic acid-based composite supramolecular gel composition, which includes ascorbic acid derivatives and polymer nanoparticles; the ascorbic acid derivative is composed of ascorbic acid and C 10 ~C 20 Alkyl acid is obtained via esterification; the outer shell of the polymer nanoparticles comprises polydopamine, and the core comprises hemoglobin and a ferroptosis inducer; in solution, the hydroxyl groups of the ascorbic acid derivative and the polydopamine shell of the polymer nanoparticles self-assemble through hydrogen bonding to form an ascorbic acid-based composite supramolecular gel. Referring to the principle described above, this ascorbic acid-based composite supramolecular gel can release and generate dehydroascorbic acid and a ferroptosis inducer in tumor cells, which synergistically trigger ferroptosis in tumor cells.

[0020] Furthermore, the ascorbic acid-based composite supramolecular gel satisfies at least one of the following characteristics:

[0021] (1) The composite supramolecular gel based on ascorbic acid has a layered stacked structure;

[0022] (2) The composite supramolecular gel based on ascorbic acid has a porous structure;

[0023] (3) The ascorbic acid-based composite supramolecular gel has a storage modulus greater than its loss modulus in the angular frequency range of 0.1 to 10 rad / s.

[0024] (4) The ascorbic acid-based composite supramolecular gel exhibits a crossover of storage modulus and loss modulus within a stress range of 30% to 50% at a constant frequency.

[0025] Thirdly, the technical solution of this application provides a method for preparing the ascorbic acid-based composite supramolecular gel described above, comprising the following steps:

[0026] The dispersion of the polymer nanoparticles is added to an organic solution containing the ascorbic acid derivative, and stirred at 60°C to 80°C for 4 to 6 minutes to obtain the ascorbic acid-based composite supramolecular gel.

[0027] In this embodiment, after the dispersion of polymer nanoparticles is added to an organic solution containing ascorbic acid derivatives, it is stirred at a suitable temperature for a suitable time to allow the polydopamine shell of the polymer nanoparticles and the hydroxyl groups of the ascorbic acid derivatives to fully react and form a composite supramolecular gel based on ascorbic acid.

[0028] Furthermore, the method for preparing the polymer nanoparticles includes the following steps:

[0029] Hemoglobin and dopamine are mixed in an aqueous solution with a pH of 8-9 to obtain an aqueous phase;

[0030] The ferroptosis inducer was dissolved in an organic solution to obtain an organic phase;

[0031] The organic phase is added to the aqueous phase and stirred at 20℃~40℃ for 2~4 hours to obtain the first nanoparticles;

[0032] The first nanoparticles were reduced with sodium dithionite to obtain the second nanoparticles.

[0033] The second nanoparticles were placed in an oxygen atmosphere for oxygenation to obtain the polymer nanoparticles.

[0034] This embodiment provides a method for preparing polymer nanoparticles. Hemoglobin and dopamine are mixed in an alkaline aqueous solution to form an aqueous phase, thereby polymerizing dopamine on the surface of hemoglobin. An ferroptosis inducer is mixed in an organic solvent to form an organic phase, which is then added to the aqueous phase and mixed. The ferroptosis inducer is then coated under stirring to form nanoparticles with a shell and core. Since hemoglobin raw materials are easily oxidized by oxygen in the air during storage and become methemoglobin, the core of the first nanoparticle is methemoglobin. After reducing the first nanoparticle with sodium dithionite solution, the methemoglobin is reduced, so the core of the second nanoparticle is ferriglobin. When oxygen is introduced into the second nanoparticle, the ferriglobin is loaded with oxygen.

[0035] Fourthly, the technical solution of this application provides the ascorbic acid-based composite supramolecular gel composition described above, or the application of the ascorbic acid-based composite supramolecular gel described above in the preparation of antitumor drugs. In this embodiment, due to the use of the ascorbic acid-based composite supramolecular gel composition or the ascorbic acid-based composite supramolecular gel described above, the obtained antitumor drug has a better antitumor effect and low cytotoxicity.

[0036] Fifthly, the technical solution of this application provides an antitumor drug formulation, including the ascorbic acid-based composite supramolecular gel and other components with antitumor activity as described above.

[0037] In this embodiment, other components with antitumor activity are encapsulated within the aforementioned ascorbic acid-based composite supramolecular gel, and their efficacy remains unaffected. This antitumor drug formulation can be used for multi-target, multi-mechanism therapies in tumor treatment; for example, it can be used in combination with immunotherapy drugs, achieving better therapeutic effects than single-therapy.

[0038] Sixthly, the technical solution of this application provides a method for preparing the antitumor drug formulation described above, comprising the following steps:

[0039] The dispersion of polymer nanoparticles and the other antitumor active ingredients are added to an organic solution containing ascorbic acid derivatives, and stirred at 60°C to 80°C for 4 to 6 minutes to obtain the antitumor drug formulation.

[0040] In this embodiment, other antitumor active ingredients are passively encapsulated during gel formation and loaded into the porous three-dimensional network structure of the gel through various forces. After the gel molecules enter the tumor microenvironment, they act on the tumor tissue as the gel relaxes and releases (see the release mechanism in the first aspect), without affecting the efficacy. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0042] Figure 1 This is a schematic diagram illustrating the preparation process and structure of an ascorbic acid-based composite supramolecular gel according to an embodiment of this application.

[0043] Figure 2 The images show physical and comparative diagrams of the ascorbic acid-based composite supramolecular gel of Example 1 of this application.

[0044] Figure 3 The image shows a scanning electron microscope image of the ascorbic acid-based composite supramolecular gel of Example 1 of this application, with a scale bar of 5 μm.

[0045] Figure 4 The curves showing the changes in storage modulus and loss modulus of the ascorbic acid-based composite supramolecular gel in Example 1 of this application, scanned in the angular frequency range of 0.1 to 10 rad / s.

[0046] Figure 5The storage modulus and loss modulus of the ascorbic acid-based composite supramolecular gel in Example 1 of this application are shown in the stress scan curves from 0.1% to 1000%.

[0047] Figure 6 The time-mass decay curves of the ascorbic acid-based composite supramolecular gel of Example 1 of this application at 0 U / mL, 1 U / mL, and 10 U / mL esterase concentrations are shown.

[0048] Figure 7 The results of glutathione determination are for the polymer nanoparticles of Example 1 of this application after co-incubation with ascorbic acid and breast cancer cells;

[0049] Figure 8 The results of reactive oxygen species determination are for the polymer nanoparticles and ascorbic acid mixture of Example 1 of this application after co-incubation with breast cancer cells;

[0050] Figure 9 The results of glutathione peroxidase 4 determination are for the polymer nanoparticles and ascorbic acid mixture of Example 1 of this application after co-incubation with breast cancer cells.

[0051] Figure 10 The statistical results show the percentage of live and dead cells after co-incubating the polymer nanoparticles and ascorbic acid mixture of Example 1 of this application with breast cancer cells;

[0052] Figure 11 The results of tumor inhibition in breast cancer model mice using ascorbic acid-based composite supramolecular gel in Example 1 of this application;

[0053] Figure 12 The results of Example 1 of this application on the inhibition of glutathione peroxidase 4 expression in tumor tissues of breast cancer model mice by ascorbic acid-based composite supramolecular gel;

[0054] Figure 13 The results of the investigation into the esterase-responsive release of the ascorbic acid derivative in Example 2 of this application;

[0055] Figure 14 The results of the treatment of mice with breast cancer after surgery using the ascorbic acid-based composite supramolecular gel in Example 2 of this application;

[0056] Figure 15 The results of tumor inhibition in mice with distal metastasis of breast cancer using ascorbic acid-based composite supramolecular gel in Example 2 of this application;

[0057] Figure 16The results of Example 2 of this application on the promotion of CD8⁺ T cell growth in mice with distant metastasis of breast cancer using ascorbic acid-based composite supramolecular gel;

[0058] Figure 17 The results of Example 2 of this application on the promotion of CD4⁺ T cell growth in mice with distant metastasis of breast cancer using ascorbic acid-based composite supramolecular gel;

[0059] Figure 18 The results of Example 2 of this application on the promotion of central memory T cell growth in mice with distant metastasis of breast cancer using ascorbic acid-based composite supramolecular gel. Detailed Implementation

[0060] The present application is further described below with reference to embodiments and examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, it should be understood that after reading the teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the protection scope of the appended claims.

[0061] 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 application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0062] the term

[0063] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0064] The term “and / or” as used herein includes any one of two or more of the related listed items, as well as any and all combinations of the related listed items, wherein any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items.

[0065] In this document, terms such as "preferred," "better," and "more preferred" are merely descriptions of implementation methods or examples that achieve better results, and should be understood as not constituting a limitation on the scope of protection of this application.

[0066] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0067] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," "fifth aspect," and "sixth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," "fifth," and "sixth" serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0068] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0069] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, optional numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0070] In this application, weight can be a well-known unit of mass in the pharmaceutical field, such as μg, mg, g, or kg.

[0071] In this application, unless otherwise specified, the terms "size", "particle size", and "diameter" generally refer to average values.

[0072] In this application, unless otherwise specified, molecular weight refers to average molecular weight, and further, unless otherwise specified, refers to weight-average molecular weight.

[0073] In this article, the abbreviations include: AA: ascorbic acid; DHA: dehydroascorbic acid; AP: ascorbyl palmitate (an ascorbic acid derivative); 4T1: breast cancer cells; aPD-1: anti-programmed death receptor 1 antibody (a biological agent used for immunotherapy).

[0074] In a first aspect, the technical solution of the embodiments of this application provides a composite supramolecular gel composition based on ascorbic acid, including ascorbic acid derivatives and polymer nanoparticles;

[0075] Ascorbic acid derivatives, through ascorbic acid and C 10 ~C 20 Alkyl acids are obtained through esterification.

[0076] The polymer nanoparticles have a core and a shell covering the core, the shell being composed of polydopamine and the core being composed of hemoglobin and ferroptosis inducer.

[0077] In the ascorbic acid-based composite supramolecular gel composition of this application embodiment, the ascorbic acid derivative is composed of ascorbic acid and C 10 ~C 20 Alkyl acids are obtained through esterification and contain ester bonds. The outer shell of the polymer nanoparticles is polydopamine, which allows the polymer nanoparticles to enter the cell through dopamine receptors. The core contains ferroptosis inducers and hemoglobin. Ferroptosis inducers can promote cell death, and hemoglobin can provide an oxygen environment. Since tumor tissue contains abundant esterases, the ascorbic acid-based composite supramolecular gel composition releases ascorbic acid and polymer nanoparticles under the action of esterases. The oxygenated hemoglobin in the polymer nanoparticles compensates for the hypoxia of the tumor microenvironment by releasing oxygen, promoting the oxidation of ascorbic acid to dehydroascorbic acid. Subsequently, dehydroascorbic acid enters the cell and triggers ferroptosis by inducing cellular oxidative stress. Since the tumor cell interior is usually acidic, when the polymer nanoparticles enter the tumor cell, the polydopamine shell of the polymer nanoparticles undergoes protonation and structural relaxation in the acidic environment, thereby releasing ferroptosis inducers. Ferroptosis inducers and dehydroascorbic acid work synergistically to further aggravate ferroptosis in tumor cells.

[0078] Furthermore, the weight ratio of ascorbic acid derivative to polymer nanoparticles is 1:(0.03~0.05).

[0079] Furthermore, the ascorbic acid derivatives include at least one of palmitic ascorbate, capric ascorbate, lauric ascorbate, myristic ascorbate, and oleic ascorbate.

[0080] Furthermore, the particle size of the polymer nanoparticles is 100 nm to 140 nm.

[0081] Furthermore, the thickness of the outer shell is 5% to 10% of the particle size of the polymer nanoparticles.

[0082] Furthermore, in polymer nanoparticles, the core accounts for ≥80% of the total mass.

[0083] Secondly, the technical solution of this application provides a composite supramolecular gel based on ascorbic acid, which is formed by the self-assembly of the composite supramolecular gel composition based on ascorbic acid in a solution environment.

[0084] The ascorbic acid-based composite supramolecular gel of this application embodiment utilizes the aforementioned ascorbic acid-based composite supramolecular gel composition, which includes ascorbic acid derivatives and polymer nanoparticles; the ascorbic acid derivative is composed of ascorbic acid and C 10 ~C 20 Alkyl acids are obtained through esterification. The outer shell of the polymer nanoparticles comprises polydopamine, while the core comprises hemoglobin and a ferroptosis inducer. In solution, the hydroxyl groups of the ascorbic acid derivative and the polydopamine shell of the polymer nanoparticles self-assemble through hydrogen bonding to form an ascorbic acid-based composite supramolecular gel. Referring to the principle described above, this ascorbic acid-based composite supramolecular gel can be degraded by esterases, releasing ascorbic acid and polymer nanoparticles. The polymer nanoparticles exhibit acid-responsive release properties, releasing the ferroptosis inducer from the core in an acidic cellular environment, thus enhancing the ferroptosis sensitivity of cells. Since tumor tissue contains abundant esterases, this ascorbic acid-based composite supramolecular gel can be degraded by esterases to release ascorbic acid and polymer nanoparticles. The oxygen carried by the hemoglobin in the polymer nanoparticles compensates for the hypoxic environment of the tumor microenvironment, allowing ascorbic acid to be oxidized to dehydroascorbic acid. Subsequently, dehydroascorbic acid triggers ferroptosis in tumor cells by inducing cellular oxidative stress. Furthermore, the ascorbic acid-based composite supramolecular gel satisfies at least one of the following characteristics:

[0085] (1) The ascorbic acid-based composite supramolecular gel has a layered stacked structure, indicating that the ascorbic acid-based composite supramolecular gel has the structural characteristics of a supramolecular gel. The ascorbic acid-based composite supramolecular gel is a gel formed through non-covalent interactions (such as hydrogen bonds, van der Waals forces, π-π stacking, etc.). Such materials usually have a dynamic and reversible structure and have better self-healing or responsiveness than traditional chemically cross-linked gels.

[0086] (2) The composite supramolecular gel based on ascorbic acid has a porous structure, indicating that the composite supramolecular gel based on ascorbic acid has the structural characteristics of a supramolecular gel.

[0087] (3) The storage modulus of the composite supramolecular gel based on ascorbic acid is greater than the loss modulus in the angular frequency range of 0.1 to 10 rad / s, indicating that the composite supramolecular gel based on ascorbic acid has elastic colloidal properties.

[0088] (4) The storage modulus and loss modulus of the composite supramolecular gel based on ascorbic acid cross within the stress range of 30% to 50% at a constant frequency. The cross between the storage modulus and loss modulus under stress of 30% to 50% means that the composite supramolecular gel based on ascorbic acid has certain stability and retention characteristics.

[0089] Thirdly, the technical solution of this application provides a method for preparing the ascorbic acid-based composite supramolecular gel described above, comprising the following steps:

[0090] A dispersion of polymer nanoparticles was added to an organic solution containing ascorbic acid derivatives and stirred at 60℃~80℃ for 4~6 minutes to obtain a composite supramolecular gel based on ascorbic acid.

[0091] In this embodiment, after the dispersion of polymer nanoparticles is added to an organic solution containing ascorbic acid derivatives, it is stirred at a suitable temperature for a suitable time to allow the polydopamine shell of the polymer nanoparticles and the hydroxyl groups of the ascorbic acid derivatives to fully react and form a composite supramolecular gel based on ascorbic acid.

[0092] Furthermore, the preparation method of polymer nanoparticles includes the following steps:

[0093] Hemoglobin and dopamine are mixed in an aqueous solution with a pH of 8-9 to obtain an aqueous phase;

[0094] The ferroptosis inducer was dissolved in an organic solution to obtain an organic phase;

[0095] The organic phase was added to the aqueous phase and stirred at 20℃~40℃ for 2~4 hours to obtain the first nanoparticles;

[0096] The first nanoparticles were reduced with dithionite solution to obtain the second nanoparticles.

[0097] The second nanoparticle was placed in an oxygen atmosphere for oxygenation to obtain polymer nanoparticles.

[0098] This embodiment provides a method for preparing polymer nanoparticles. Hemoglobin and dopamine are mixed in an alkaline aqueous solution to form an aqueous phase, thereby polymerizing dopamine on the surface of hemoglobin. An ferroptosis inducer is mixed in an organic solvent to form an organic phase, which is then added to the aqueous phase and mixed. The ferroptosis inducer is then coated under stirring to form nanoparticles with a shell and core. Since hemoglobin raw materials are easily oxidized by oxygen in the air during storage and become methemoglobin, the core of the first nanoparticle is methemoglobin. After reducing the first nanoparticle with sodium dithionite solution, the methemoglobin is reduced, so the core of the second nanoparticle is ferriglobin. When oxygen is introduced into the second nanoparticle, the ferriglobin is loaded with oxygen.

[0099] Fourthly, the technical solutions of this application provide the above-described ascorbic acid-based composite supramolecular gel composition, or the above-described ascorbic acid-based composite supramolecular gel in the preparation of antitumor drugs.

[0100] In this embodiment, due to the use of the ascorbic acid-based composite supramolecular gel composition or the ascorbic acid-based composite supramolecular gel described above, the obtained antitumor drug has a better antitumor effect and low cytotoxicity.

[0101] Fifthly, the technical solution of this application provides an anti-tumor drug formulation, the composition of which includes the ascorbic acid-based composite supramolecular gel mentioned above and other components with anti-tumor activity. Specific examples of other components with anti-tumor activity include aPD-1 (i.e., anti-PD-1 antibody), which can activate immune checkpoint inhibitors that attack tumors by blocking the PD-1 / PD-L1 signaling pathway.

[0102] In some embodiments, in the antitumor drug formulation, other components with antitumor activity are passively encapsulated during the formation of the ascorbic acid-based composite supramolecular gel and are trapped in the network pores of the ascorbic acid-based composite supramolecular gel.

[0103] Sixthly, the technical solution of this application provides a method for preparing an antitumor drug formulation, comprising the following steps:

[0104] The dispersion of polymer nanoparticles and other components with antitumor activity are added to an organic solution containing ascorbic acid derivatives, and stirred at 60℃~80℃ for 4~6 minutes to obtain an antitumor drug formulation.

[0105] The other antitumor active ingredients mentioned above refer to those other than ascorbic acid and ferroptosis-inducing inhibitors.

[0106] The following are some specific examples.

[0107] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this application document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0108] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.

[0109] Example 1

[0110] 1.1 Preparation of polymer nanoparticles

[0111] Hb (3 mg) and dopamine (0.6 mg) were dissolved in Tris-buffer (7.2 mL, pH 8.5) to obtain the aqueous phase. Lovastatin (Lv), fluvastatin (Flu), sorafenib (Sor), sulfasalazine (Sas), temozolomide (Tmz), and dexamethasone (Dex) (0.4 mg) were dissolved in DMSO (0.8 mL) to obtain the organic phase. The organic phase was then added dropwise to the aqueous phase, and the mixture was stirred at room temperature for 3 h. After the reaction was completed, the resulting mixture was subjected to ultrafiltration to remove the alkaline reaction medium and terminate the reaction. The ultrafiltration product was then washed with PBS and resuspended in 1 mL.

[0112] The resuspended solution was reduced by adding 100 μL of sodium dithionite solution (400 μg / mL) to convert mHb to Hb. Finally, the reduced mixture was subjected to ultrafiltration to remove excess sodium dithionite and resuspended in 1 mL of PBS. After continuous bubbling with O2, polymer nanoparticles were obtained.

[0113] The particle size and polydispersity index of each group of nanoparticles were determined using a particle size analyzer, and the results are shown in Table 1. In Table 1, Lv / Hb-PDA refers to the polymer nanoparticles whose core is lovastatin (Lv) and hemoglobin (Hb), and whose shell is polydopamine (PDA), and so on.

[0114] Table 1. Particle size distribution and dispersion coefficient of drug-loaded hemoglobin polydopamine nanoparticles

[0115]

[0116] Polymer nanoparticles Hb-PDA were prepared, with the core of the polymer nanoparticles being hemoglobin (Hb) and the shell being polydopamine (PDA). The preparation method was similar to that of Lv / Hb-PDA, except that Hb (3 mg) and dopamine (0.6 mg) were dissolved in Tris-buffer (7.2 mL, pH 8.5) to obtain an aqueous phase. No organic phase was added, and the aqueous phase was directly stirred and reacted at room temperature for 3 h.

[0117] Polymer nanoparticles mHb-PDA were prepared, with the core of the polymer nanoparticles being hemoglobin (mHb) and the shell being polydopamine (PDA). The preparation method was similar to that of Lv / Hb-PDA, except that after the reaction, the resulting mixture solution was subjected to ultrafiltration to remove the alkaline reaction medium to terminate the reaction. Subsequently, the ultrafiltration product was washed with PBS and resuspended to 1 mL to obtain the homogeneous nanoparticles, omitting the reduction and oxygenation treatment steps.

[0118] 1.2 Preparation of ascorbic acid-based composite supramolecular gel

[0119] Taking Lv / Hb-PDA as an example, the specific steps for preparing an ascorbic acid-based composite supramolecular gel are provided: Lv / Hb-PDA (800 μL, 2 mg / mL) is added dropwise to DMSO (200 μL) containing AP (40 mg), and the mixture is vortexed until homogeneous. The resulting mixture is heated in a 70 ℃ water bath for 5 min and then cooled to room temperature to form a gel. Finally, the gel is washed three times with PBS to obtain the ascorbic acid-based composite supramolecular gel Lv / HPAGel. Some of the raw materials and products in the above process are shown in the following figures. Figure 2 AP forms a gel under the action of Lv / Hb-PDA, but does not form a gel in aqueous solution, indicating that the gel is formed through the co-assembly of AP and Lv / Hb-PDA.

[0120] The composite supramolecular gel HPAGel was prepared using a similar method to Lv / HPAGel, except that the polymer nanoparticles used were Hb-PDA.

[0121] 1.3 Morphology and physical properties of ascorbic acid-based composite supramolecular gels

[0122] After swelling treatment with Lv / HPAGel for 48 h, the results were observed using a scanning electron microscope. Figure 3 As shown (scale bar is 5 μm), Lv / HPAGel exhibits typical morphological characteristics of supramolecular gel layered stacking and porous microstructure.

[0123] The Lv / HPAGel was characterized using a rheometer, and the frequency sweep results are as follows: Figure 4 As shown, within the angular frequency range of 0.1–10 rad / s, the storage modulus (G') is consistently higher than the loss modulus (G"), characterizing the gel as having elastic colloidal properties; strain scanning results are as follows. Figure 5 As shown, at a constant frequency, G' and G” cross under 40% stress, indicating that Lv / HPAGel can still maintain a stable network structure under strong shear stress.

[0124] Lv / HPAGel gels were placed in media containing different concentrations of esterase and incubated at 37 °C. Gel mass was measured at specified times to assess esterase degradation characteristics. Results are as follows: Figure 6 As shown, the degree of Lv / HPAGel mass decay is positively correlated with the esterase concentration.

[0125] The above experimental results demonstrate that Lv / HPAGel possesses typical microstructure and rheological characteristics of supramolecular gels and can be degraded by esterases.

[0126] 1.4 Antitumor activity of ascorbic acid-based composite supramolecular gel compositions and ascorbic acid-based composite supramolecular gels

[0127] 1.4.1 Activity inducing oxidative stress and ferroptosis in breast cancer cells

[0128] 4T1 cells were fed at a rate of 2 × 10 5 The cells were seeded at a density of cells / well in 12-well plates. The control group was incubated for 12 hours under hypoxic conditions without the addition of reagents. The AA group, Hb-PDA group, mHb-PDA+AA group, and Hb-PDA+AA group were incubated for 12 hours under hypoxic conditions with a mixture of AA (200 μg / mL), Hb-PDA (100 μg / mL), mHb-PDA (100 μg / mL) and AA (200 μg / mL), and a mixture of Hb-PDA (100 μg / mL) and AA (200 μg / mL), respectively.

[0129] Glutathione (GSH) assay: Cells were collected after incubation, and the total protein concentration was determined using the BCA method. Intracellular GSH levels were measured using a reduced glutathione (GSH) assay kit. Finally, the absorbance was measured using a microplate reader, and the relative concentration of GSH was calculated. Results are as follows: Figure 7 As shown, under hypoxic conditions, cells co-incubated with Hb-PDA and AA can significantly reduce intracellular GSH levels.

[0130] Measurement of reactive oxygen species (ROS): After cell incubation, ROS probes were loaded, and intracellular ROS levels were measured using flow cytometry. Results are as follows: Figure 8 As shown, under hypoxic conditions, cells co-incubated with Hb-PDA and AA can induce a significant accumulation of intracellular ROS.

[0131] Glutathione peroxidase 4 (GPX4) assay: 4T1 cells were incubated at 4 × 10⁻⁶ cells per day. 5 Cells were seeded at a density of [number] cells / well in 6-well plates. The control group (no reagents added) was incubated under hypoxic conditions for 24 hours. The Hb-PDA + AA group, Lv / Hb-PDA group, and Lv / Hb-PDA + AA group were incubated under hypoxic conditions with a mixture of Hb-PDA (100 μg / mL) and AA (200 μg / mL), Lv / Hb-PDA (100 μg / mL), and Lv / Hb-PDA (100 μg / mL) and AA (200 μg / mL), respectively, for 24 hours. After incubation, cells were collected, and the expression level of GPX4 was detected by Western blotting. The results are as follows: Figure 9 As shown, cells co-incubated with Hb-PDA and AA significantly reduced the expression of intracellular GPX4.

[0132] The above experimental results demonstrate that co-incubation of cells with Hb-PDA and AA can induce intracellular GSH consumption and ROS accumulation, leading to intracellular oxidative stress. The downregulation of intracellular GPX4 expression also proves the occurrence of ferroptosis.

[0133] Cell viability / deadness assay: 4T1 cells were stained at 1×10⁻⁶. 5 Cells were seeded at a density of cells / well in confocal culture dishes. The control group (without reagents) was incubated under hypoxic conditions for 24 hours. The Hb-PDA + AA group, Lv / Hb-PDA group, and Lv / Hb-PDA + AA group were incubated under hypoxic conditions with a mixture of Hb-PDA (100 μg / mL) and AA (200 μg / mL), Lv / Hb-PDA (100 μg / mL), and Lv / Hb-PDA (100 μg / mL) and AA (200 μg / mL), respectively, for 24 hours. After incubation, cells were collected, and live cells were labeled with Calcein-AM green fluorescence, while dead cells were labeled with PI red fluorescence. The distribution of live / dead cells was observed using laser confocal microscopy. Results are as follows: Figure 10 As shown, cells co-incubated with Lv / Hb-PDA and AA were able to induce more than 90% cell death, which was significantly higher than in other groups.

[0134] 1.4.2 Activity in treating breast cancer model mice

[0135] 4T1 breast cancer cells were resuspended in a mixture of basal culture medium and Matrigel (3:1) at 1×10⁻⁶ ppm. 6 In vitro mammary cancer mouse model was established by inoculating the right mammary fat pad of mice (Balb / c, female, 5 weeks old, 20g) with a number of 1 / 2 individuals. Tumors were then inoculated into the fat pad of the right mammary gland. When the tumor volume approached 0.1 cm², the tumor was considered a target. 3 Mice bearing tumors were randomly divided into four groups and injected in situ with saline (Saline group), a mixture of Lv / Hb-PDA and AA (Lv / Hb-PDA+AA group), HPAGel (HPAGel group), and Lv / HPAGel (Lv / HPAGel group), respectively. The dosages of Lv and AA were 2.1 mg / kg and 127.5 mg / kg, respectively. During treatment, the long and short diameters of the tumor were measured every two days, and tumor volume was calculated to monitor changes in tumor volume. Results are as follows: Figure 11As shown, compared to the Lv / Hb-PDA and AA mixture and the drug-free gel group, Lv / HPAGel exhibited a significant tumor-suppressive effect. For the investigation of ferroptosis, GPX4 in tumor tissue was detected by immunohistochemistry after tumor tissue collection, and the expression of GPX4 in the tumor was determined using a fully automated slide scanning system. The results are as follows... Figure 12 As shown (scale bar is 50 μm), Lv / HPAGel can significantly reduce the expression of GPX4 in tumors and enhance anti-tumor efficacy by promoting tumor ferroptosis.

[0136] Example 2

[0137] 2.1 Preparation of polymer nanoparticles Lv / Hb-PDA

[0138] Hb (3 mg) and dopamine (0.6 mg) were dissolved in Tris-buffer (7.2 mL, pH 8.5), and Lv (0.4 mg) was dissolved in DMSO (0.8 mL). The organic phase was then added dropwise to the aqueous phase, and the mixture was stirred at room temperature for 3 h. After the reaction was complete, the resulting mixture was subjected to ultrafiltration to remove the alkaline reaction medium and terminate the reaction. The ultrafiltration product was then rinsed with PBS and resuspended to 1 mL. The resuspended solution was reduced by adding 100 μL of sodium dithionite solution (400 μg / mL) to convert mHb to Hb. Finally, the reduced mixture was subjected to ultrafiltration to remove excess sodium dithionite and resuspended in 1 mL of PBS. After continuous O2 bubbling, Lv / Hb-PDA was obtained.

[0139] 2.2 Preparation of ascorbic acid-based composite supramolecular gel aPD-1@Lv / HPAGel

[0140] The Lv / Hb-PDA (800 μL, 2 mg / mL) and aPD-1 solution (72 μL, 18.5 mg / mL) were mixed and then added to DMSO (200 μL) containing AP (40 mg). The mixture was vortexed until homogeneous. The resulting mixture was heated in a water bath at 70 °C for 5 minutes and then cooled to room temperature to form a gel. Finally, the gel was washed three times with PBS to obtain aPD-1@Lv / HPAGel.

[0141] 2.3 Investigation of the responsive release of aPD-1 esterase

[0142] Alexa Fluor 488-labeled aPD-1 (0.2 μg / mL) was mixed with Lv / Hb-PDA (2 mg / mL), and then the mixture (800 μL) was added dropwise to DMSO (200 μL) containing AP (40 mg), vortexing until homogeneous. The resulting mixture was heated in a 70°C water bath for 5 minutes, then cooled to form a gel, and finally washed three times with PBS. Subsequently, the fluorescently labeled gel (100 μL) was added to release media containing different concentrations of esterases and incubated at 37°C. The supernatant was collected at specified time points, and its fluorescence value was measured using a microplate reader. The results are as follows. Figure 13 As shown, the release of aPD-1 is directly proportional to the concentration of esterase, proving that aPD-1 can be successfully released from the gel under the action of esterase.

[0143] 2.4 Evaluation of the effect of composite supramolecular gel combined with immunotherapy on the postoperative treatment of breast cancer

[0144] Mouse-derived 4T1 cells were resuspended in a mixture of basal medium and Matrigel (3:1) at a concentration of 1×10⁻⁶. 6 Inoculate the right mammary fat pad of mice (Balb / c, female, 5 weeks old, 20 g) with a dose of 120 ... 3 At the time of surgery, 90% of the tumor volume was surgically removed to establish a tumor model, and mice were randomly divided into four groups. On the second day after surgery, mice were injected in situ with saline (Saline group), anti-PD-1 antibody (aPD-1 group), supramolecular gel loaded with Lv (Lv / HPAGel group), and supramolecular gel loaded with both aPD-1 and Lv (aPD-1@Lv / HPAGel group), respectively. The dosages of aPD-1, Lv, and AA were 10 mg / kg, 2.1 mg / kg, and 127.5 mg / kg, respectively. Tumor growth was monitored in mice during drug treatment. Results are as follows: Figure 14 As shown, compared with aPD-1 monotherapy and other drug administration groups, aPD-1@Lv / HPAGel can significantly enhance the treatment effect of breast cancer.

[0145] 2.5 Evaluation of aPD-1@Lv / HPAGel immunotherapy in the treatment of distant metastatic breast cancer

[0146] Mouse-derived 4T1 cells were resuspended in a mixture of basal medium and Matrigel (3:1) at 1×10⁻⁶ ppm. 6 In vitro mammary cancer mouse model was established by inoculating the right mammary fat pad of mice (Balb / c, female, 5 weeks old, 20g) with a number of 1 / 2 individuals. Tumors were implanted into the fat pads of the right mammary glands of mice. The tumors were cultured until they reached a size of 0.1 cm². 3At that time, tumor-bearing mice were randomly divided into four groups and injected in situ with saline, anti-PD-1 antibody (aPD-1), supramolecular gel loaded with Lv (Lv / HPAGel), and supramolecular gel loaded with both aPD-1 and Lv (aPD-1@Lv / HPAGel), respectively. The dosages of aPD-1, Lv, and AA were 10 mg / kg, 2.1 mg / kg, and 127.5 mg / kg, respectively. On the sixth day after administration, the mice were further treated with 8 × 10⁸ doses of saline, anti-PD-1 antibody (aPD-1), Lv, and AA. 5 A number of mice per left mammary fat pad were inoculated to establish a distant metastasis model. Tumor growth was monitored in mice during drug treatment. Results are as follows: Figure 15 As shown, compared with aPD-1 monotherapy and other drug administration groups, aPD-1@Lv / HPAGel effectively inhibited distant tumor metastasis. After treatment, mouse lymph nodes and spleens were collected and prepared into single-cell suspensions. Flow cytometry was used to measure the levels of T cell-related factors CD4 and CD8, as well as the levels of central memory T cell-related factors CD62L and CD44. The results, as shown in Figures 16-18, significantly increased the ratio of CD4 T cells to CD8 T cells in lymph nodes and the proportion of central memory T cells in the spleen, demonstrating that the combination of aPD-1@Lv / HPAGel and immune checkpoint blockade therapy can significantly enhance the body's immunity.

[0147] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0148] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0149] The embodiments described above merely illustrate several implementation methods of this application and should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Furthermore, it should be understood that after reading the above teachings, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A composite supramolecular gel composition based on ascorbic acid, characterized in that, Including ascorbic acid derivatives and polymer nanoparticles; The ascorbic acid derivative is obtained through ascorbic acid and C. 10 ~C 20 Alkyl acids are obtained through esterification. The polymer nanoparticles have a core and a shell covering the core, the shell comprising polydopamine and the core comprising hemoglobin and ferroptosis inducer.

2. The ascorbic acid-based composite supramolecular gel composition according to claim 1, characterized in that, The weight ratio of the ascorbic acid derivative to the polymer nanoparticles is 1:(0.03~0.05); and / or, The ascorbic acid derivatives include at least one of palmitic ascorbate, capric ascorbate, lauric ascorbate, myristic ascorbate, and oleic ascorbate.

3. The ascorbic acid-based composite supramolecular gel composition according to claim 1 or 2, characterized in that, At least one of the following conditions must be met: (1) The particle size of the polymer nanoparticles is 100 nm to 140 nm; (2) The thickness of the outer shell is 5% to 10% of the particle size of the polymer nanoparticles; (3) In the polymer nanoparticles, the mass percentage of the core is ≥80%.

4. A composite supramolecular gel based on ascorbic acid, characterized in that, The ascorbic acid-based composite supramolecular gel composition according to any one of claims 1 to 3 is formed by self-assembly in a solution environment.

5. The ascorbic acid-based composite supramolecular gel according to claim 4, characterized in that, The ascorbic acid-based composite supramolecular gel satisfies at least one of the following characteristics: (1) The composite supramolecular gel based on ascorbic acid has a layered stacked structure; (2) The composite supramolecular gel based on ascorbic acid has a porous structure; (3) The ascorbic acid-based composite supramolecular gel has a storage modulus greater than its loss modulus in the angular frequency range of 0.1 to 10 rad / s. (4) The ascorbic acid-based composite supramolecular gel exhibits a crossover of storage modulus and loss modulus within a stress range of 30% to 50% at a constant frequency.

6. A method for preparing the ascorbic acid-based composite supramolecular gel according to claim 4 or 5, characterized in that, Includes the following steps: The dispersion of the polymer nanoparticles is added to an organic solution containing the ascorbic acid derivative, and stirred at 60°C to 80°C for 4 to 6 minutes to obtain the ascorbic acid-based composite supramolecular gel.

7. The preparation method according to claim 6, characterized in that, The preparation method of the polymer nanoparticles includes the following steps: Hemoglobin and dopamine are mixed in an aqueous solution with a pH of 8-9 to obtain an aqueous phase; The ferroptosis inducer was dissolved in an organic solution to obtain an organic phase; The organic phase is added to the aqueous phase and stirred at 20℃~40℃ for 2~4 hours to obtain the first nanoparticles; The first nanoparticles were reduced with sodium dithionite to obtain the second nanoparticles. The second nanoparticles were placed in an oxygen atmosphere for oxygenation to obtain the polymer nanoparticles.

8. The use of an ascorbic acid-based composite supramolecular gel composition according to any one of claims 1 to 3, or the use of an ascorbic acid-based composite supramolecular gel according to claim 4 or 5, in the preparation of antitumor drugs.

9. An antitumor drug formulation, characterized in that, It includes the ascorbic acid-based composite supramolecular gel as described in claim 4 or 5 and other components with antitumor activity.

10. A method for preparing an antitumor drug formulation according to claim 9, characterized in that, Includes the following steps: The dispersion of polymer nanoparticles and the other antitumor active ingredients are added to an organic solution containing ascorbic acid derivatives, and stirred at 60°C to 80°C for 4 to 6 minutes to obtain the antitumor drug formulation.

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