WO2024041535A1

WO2024041535A1 - Nano-composition, preparation method therefor, and use thereof

Info

Publication number: WO2024041535A1
Application number: PCT/CN2023/114266
Authority: WO
Inventors: 石凯; 毕洪书
Original assignee: 赣州和美药业股份有限公司
Priority date: 2022-08-22
Filing date: 2023-08-22
Publication date: 2024-02-29
Also published as: WO2024041532A1; CN117603299A; CN117599013A

Abstract

The present disclosure relates to a nano-composition, a preparation method therefor, and use thereof. The nano-composition is formed by self-assembly of an oligopeptide containing a tryptophan dipeptide motif and cisplatin. The general formula of the oligopeptide is Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-W-W-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12, wherein W is tryptophan, and Xaa1-Xaa12 are each independently absent or each independently represent an arbitrary amino acid residue. The nano-composition features mild preparation conditions, simple operation, environmental friendliness, etc., thus being suitable for industrial production and enabling efficient loading of medicaments. The nano-composition can be used for the preparation of anti-tumor medicaments.

Description

Nanocompositions and preparation methods and uses thereof

Cross-references to related applications

This application claims priority to Chinese patent applications No. 202211006334.0 and No. 202211005108.0, which were submitted to the China Patent Office on August 22, 2022. The disclosure contents of the said Chinese patent applications are incorporated herein by reference in their entirety. For All purposes.

field

The present disclosure relates to the field of biomedicine, and in particular to self-assembled nanocompositions formed from oligopeptides containing tryptophan dipeptide groups and cisplatin and preparation methods and uses thereof.

background

With the widespread application of nanotechnology in the field of biomedicine, nanocarrier systems based on various polymers, inorganic or lipid materials have shown great potential application value and development prospects in achieving effective delivery of drugs in the body. Compared with free drug solutions, nanocarrier systems have the advantages of improving drug in vivo processes, adjusting drug release rates, enhancing tumor tissue targeting, improving bioavailability, and reducing drug toxic and side effects.

However, due to the small size and large specific surface area of nanocarrier systems constructed based on traditional materials, a larger proportion of carrier materials is usually required to achieve complete encapsulation of clinically effective therapeutic doses of drugs (especially chemical small molecule drugs), resulting in a large proportion of carrier materials. The drug dosage is low (<10%), the preparation process is complex, and it lacks reproducibility and controllability, making it difficult to achieve large-scale production and clinical transformation. In addition, the continuous introduction of large amounts of polymer or inorganic carrier materials into the body will inevitably lead to accumulated toxicity due to slow degradation or toxic metabolites. In recent years, cationic lipid nanocarriers have become popular in the delivery of nucleic acid drugs due to their good biodegradability and preparation reproducibility. However, there are still problems such as low cell transfection efficiency and certain cytotoxicity, which makes them difficult to use in certain applications. affect its use in clinical treatment.

Cisplatin, also known as cis-dichlorodiamine platinum, is a platinum-containing anti-cancer drug. It is clinically used for ovarian cancer, prostate cancer, testicular cancer, lung cancer, nasopharyngeal cancer, esophageal cancer, malignant lymphoma, Various solid tumors such as head and neck squamous cell carcinoma, thyroid cancer and osteosarcoma can show curative effect. However, cisplatin has certain toxicity when used to treat cancer and can cause side effects.

Therefore, there is an urgent need to develop new biocompatible carriers in the field of biomedicine to promote the large-scale production and clinical transformation of cisplatin-based nanomedicines.

Overview

In order to solve the above technical problems, the present disclosure provides tryptophan dipeptide-based self-assembled nanocomposites The invention discloses a substance and its preparation method and use, that is, using an indole ring with an aromatic electronic structure provided by a tryptophan dipeptide and a drug (for example, cisplatin) to drive self-assembly to form nanoparticles through π-π interactions. This construction method is different from the traditional physical encapsulation of carriers, which can achieve efficient loading of drugs and avoid the impact of covalent cross-linking on drug activity; at the same time, tryptophan peptide motifs can promote the endosomal escape and release of drugs. Efficient accumulation in the cytoplasm.

In one aspect, the present disclosure provides nanocompositions formed from the self-assembly of a tryptophan-containing oligopeptide and cisplatin.

In some embodiments, the general formula of the oligopeptide can be Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-W-W-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12, where W is tryptophan; and Xaa1- Xaa12 may each independently be absent or represent any amino acid residue.

In some embodiments, the oligopeptide can be selected from the group consisting of tryptophan-tryptophan, tryptophan-tryptophan-aspartate, tryptophan-tryptophan-arginine-glycine-aspartate Acid, tryptophan-tryptophan-arginine, tryptophan-tryptophan-glutamic acid, tryptophan-tryptophan-glycine, tryptophan-tryptophan-serine, tryptophan -Tryptophan-histidine, tryptophan-tryptophan-lysine, tryptophan-tryptophan-tyrosine, tryptophan-tryptophan-cysteine, tryptophan- Tryptophan-threonine, preferably, the oligopeptide can be selected from tryptophan-tryptophan, tryptophan-tryptophan-aspartic acid, tryptophan-tryptophan-arginine -Glycine-aspartic acid, tryptophan-tryptophan-arginine, tryptophan-tryptophan-glutamic acid.

In some embodiments, the nanocomposition can be tryptophan-tryptophan-cisplatin, tryptophan-tryptophan-aspartate-cisplatin, tryptophan-tryptophan-arginine Acid-glycine-aspartic acid-cisplatin, tryptophan-tryptophan-arginine-cisplatin or tryptophan-tryptophan-glutamic acid-cisplatin; preferably, the nanocomposition It can be tryptophan-tryptophan-glutamic acid-cisplatin.

In some embodiments, the drug loading of the nanocomposition may be from about 20% to about 60%, preferably from about 24% to about 55%, and more preferably from about 24% to about 50%.

In some embodiments, the particle size of the nanocomposition may be 10-1000 nm, preferably 20-200 nm.

In another aspect, the present disclosure provides a pharmaceutical composition comprising the above-described nanocomposition and at least one pharmaceutically acceptable excipient.

In some embodiments, the pharmaceutically acceptable excipients are selected from the group consisting of binders, fillers, disintegrants, lubricants, solvents, preservatives, antioxidants, flavoring agents, fragrances, co-solvents , one or more of emulsifiers, solubilizers, osmotic pressure regulators and colorants.

In another aspect, the present disclosure provides a method for preparing the above-mentioned nanocomposition, including: dispersing the oligopeptide in the aqueous phase C, then adding cisplatin to obtain a mixed liquid C, stirring the mixed liquid C at high speed in the dark, and removing solvent to obtain a colloidal solution of the nanocomposition.

In some embodiments, the molar ratio of the oligopeptide to cisplatin can be about 0.25-20:1, preferably about 0.5-20:1, preferably about 1-5:1, more preferably about 1-2 :1.

In some embodiments, the encapsulation efficiency of the nanocomposition may be from about 35% to about 99%, preferably from about 67.5% to about 99%, and more preferably from about 80% to about 99%.

In some embodiments, the aqueous phase C is selected from purified water, water for injection, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer, tris buffer or phosphate buffered saline (PBS) buffer; preferably, the aqueous phase C is a HEPES buffer; more preferably, the pH of the aqueous phase C is 5-9.

In some embodiments, the method further includes the step of adding a dispersant.

In some embodiments, the dispersing agent is selected from mPEG2K-NHS, serum albumin, phospholipid polyethylene glycol (depe-peg2000), lecithin, polyethylene glycol polylactic acid-co-glycolic acid (plga-peg) One or more of them; preferably, the dispersant is mPEG2K-NHS.

In some embodiments, after dispersing the oligopeptide in aqueous phase C, a step of dialysis in aqueous phase D is further included.

In some embodiments, the aqueous phase D is selected from purified water, water for injection, 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer, tris buffer or phosphate buffered saline (PBS) buffer solution; preferably, the aqueous phase D is water for injection.

In some embodiments, the mixture C is stirred at high speed at 40-60°C, preferably at 50°C in the dark.

In some embodiments, the solvent in the mixed liquid C is removed by vacuum evaporation, high-speed centrifugation, dialysis or ultrafiltration, and preferably the vacuum evaporation method is used.

In some embodiments, the method may further include the steps of adding a lyoprotectant and freeze-drying to obtain a solid powder.

In some embodiments, the lyoprotectant may be selected from one or more of sucrose, trehalose and mannitol; preferably, the lyoprotectant may be sucrose.

In another aspect, the present disclosure provides the use of the above-mentioned nanocomposition in the preparation of anti-tumor drugs.

In yet another aspect, the present disclosure provides the use of the above-mentioned nanocomposition in the preparation of a medicament for treating tumors.

In another aspect, the present disclosure provides the above-mentioned nanocomposition or the above-mentioned pharmaceutical composition for treating tumors.

In yet another aspect, the present disclosure provides a method of treating tumors, the method comprising administering to a subject in need a medicament comprising the above-mentioned nanocomposition or the above-mentioned pharmaceutical composition, such as a therapeutically effective amount of a medicament comprising the above-mentioned nanocomposition. Or the above pharmaceutical composition.

In some embodiments, the tumor is cancer.

In some embodiments, the tumor is lung cancer, breast cancer, gastric cancer, esophageal cancer, adrenocortical cancer, cutaneous squamous cell carcinoma, head and neck cancer, thyroid cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, colorectal cancer, ovarian cancer , cervical cancer, endometrial cancer, vaginal squamous cell carcinoma, testicular cancer, prostate cancer, bladder cancer, urothelial cancer, melanoma, osteosarcoma, malignant lymphoma or neuroblastoma.

Description of drawings

The accompanying drawings are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this disclosure, and thus together with the related description serve to explain the concepts of the disclosure. In the attached picture:

Figure 1 is an ESI-MS diagram of WWE prepared in Example 1;

Figure 2 is a ¹ H NMR pattern of WWE prepared in Example 1;

Figure 3 is a transmission electron microscope photograph of the P-DDP nanocomposition prepared in Example 1;

Figure 4 is a particle size distribution diagram of the P-DDP nanocomposition prepared in Example 1;

Figure 5 shows the release curve of the P-DDP nanocomposition in PBS and cell lysate in Example 3;

Figure 6 shows the particle size changes of P-DDP nanocomposition in 10% FBS and HEPES in Example 4;

Figure 7 shows the in vitro cytotoxicity of P-DDP nanocomposition and CDDP free cisplatin on SKOV3 cells in Example 5;

Figure 8 shows the in vitro cytotoxicity of P-DDP nanocomposition and CDDP free cisplatin on A549 cells in Example 5;

Figure 9 shows the effect of physiological saline, 4 mg/kg CDDP, 2 mg/kg P-DDP, 4 mg/kg P-DDP and 6 mg/kg P-DDP on tumor volume during the administration period in Example 6;

Figure 10 shows the effect of physiological saline, 4 mg/kg CDDP, 2 mg/kg P-DDP, 4 mg/kg P-DDP and 6 mg/kg P-DDP on tumor weight during the administration period in Example 6;

Figure 11 is the blood concentration-time curve of CDDP and P-DDP after administration in Example 7;

Figure 12 shows the tissue distribution of CDDP and P-DDP after administration in Example 8;

Figure 13 shows the effects of physiological saline, 4 mg/kg CDDP, 2 mg/kg P-DDP, 4 mg/kg P-DDP and 6 mg/kg P-DDP on the body weight of mice during the administration period in Example 9; and

Figure 14 shows the histopathological examination results after 20 days of administration of physiological saline, 4 mg/kg CDDP and 4 mg/kg P-DDP in Example 9.

Detailed description of the invention

The present disclosure will now be described more fully below. This disclosure may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

Furthermore, when used in the specification and appended claims of this application, the following terms have the meanings indicated unless specified to the contrary.

As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. For example, "A and/or B" may be understood to mean "A, B, or A and B." The terms "and" and "or" may be used in the sense of a conjunction or an opposite conjunction and may be understood to be equivalent to "and/or".

As used herein, the term "about" includes a stated value and means that the recited value is within a reasonable range of the stated value as determined by one of ordinary skill in the art taking into account the relevant measurements and errors associated with the measurement of the recited quantity (i.e., the limitations of the measurement system). within the acceptable deviation range. For example, "about" may mean within one or more standard deviations, or within ±20%, ±10%, or ±5% of a stated value.

It should be understood that the terms "includes," "includes," "has," "contains" and the like are intended to indicate, but not exclude, the presence of specified features, numbers, steps, operations, elements, ingredients, or combinations thereof in the present disclosure. The presence or addition of one or more other features, numbers, steps, operations, elements, components, or combinations thereof.

Reference throughout this specification to "in an embodiment," "in one embodiment," "in another embodiment," or "in some embodiments" means that at least one embodiment includes Relevant specific reference elements, structures or features described in the embodiments. Thus, appearances of the phrases "in an embodiment," "in one embodiment," "in another embodiment," or "in some embodiments" in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, specific elements, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

Unless otherwise defined herein, all terms (including technical and scientific terms) used have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The term "self-assembled nanocomposition" or "nanocomposition" as used herein refers to a nanoscale drug delivery system that self-assembles from an oligopeptide containing a tryptophan dipeptide moiety and a drug (eg, cisplatin). Oligopeptides and drugs can drive self-assembly through π-π interactions.

The term "oligopeptide" as used herein refers to an amino acid chain formed by the condensation of 2-14 amino acids with one another. In some embodiments, the oligopeptide is an amino acid chain formed from 2-10, 2-8, 2-6, 2-5, 2-4 or 2-3 amino acids condensed with each other.

The term "amino acid" as used herein refers to the main units that make up proteins in living organisms, such as glycine (G), alanine (A), valine (V), leucine (L), isoleucine Acid (I), phenylalanine (F), proline (P), serine (S), threonine (T), histidine (H), tryptophan (W), cysteine (C), aspartic acid (D), glutamic acid (E), lysine (K), tyrosine (Y), methionine (M), asparagine (N), glutamic acid Aminoamide (Q), arginine (R), etc. When describing the structure of peptides, this disclosure uses the international standard single-letter abbreviations for amino acids. Unless expressly stated otherwise, amino acids in this application refer to natural amino acids. Unless expressly stated otherwise, when referring to these amino acids, the L- and D-forms may be included.

The term "encapsulation rate" as used herein refers to the ratio of the amount of drug in the nanocomposition of the present application to the amount of drug used in the preparation (ie, the dosage).

The term "drug loading" as used herein refers to the ratio of the amount of drug to the sum of the amount of drug and the amount of oligopeptide in the nanocomposition of the present application.

The term "pharmaceutically acceptable excipients" as used herein refers to add-ons other than the main drug in pharmaceutical preparations that are approved by the national drug regulatory agency as acceptable for use in humans or livestock, and may also be called excipients. For example, binders, fillers, disintegrants, and lubricants in tablets; matrix parts in semi-solid preparations; solvents, preservatives, antioxidants, flavorings, aromatics, etc. in liquid preparations. Cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc.

The term "subject" may include humans and domestic animals, such as laboratory animals and household pets (eg, cats, dogs, pigs, cattle, sheep, goats, horses, rabbits), as well as non-domesticated animals, such as wild animals, and the like.

The term "therapeutically effective amount" as used herein refers to an amount of the nanocomposition of the present application that is sufficient to effect treatment of tumors in a mammal, preferably a human, when administered to a mammal, preferably a human. The amount of the nanocomposition of the present application that constitutes a "therapeutically effective amount" will vary depending on the active drug, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one of ordinary skill in the art. based on their own knowledge and the contents of this disclosure.

The term "treatment" as used herein encompasses the treatment of a tumor in a mammal, preferably a human, suffering from a tumor and includes:

(i) Prevent the development of tumors in mammals, especially when these mammals are susceptible to tumors but have not yet been diagnosed with tumors;

(ii) inhibit tumors, that is, prevent their development;

(iii) relieve the tumor, i.e. cause the regression of the tumor; or

(iv) Relieve symptoms caused by tumors.

Nanocomposition

The present disclosure provides nanocompositions formed by self-assembly of oligopeptides containing tryptophan dipeptide motifs and drugs, wherein the oligopeptides and drugs are connected through π-π interactions.

The general formula of the oligopeptide can be Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-W-W-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12, where W is tryptophan; Xaa1-Xaa12 can each be independently absent. or represents any amino acid residue.

In some embodiments, Xaa1-Xaa12 may each be the same or different. In some embodiments, If Xaa1-Xaa12 are not present at the same time, the oligopeptide used is tryptophan-tryptophan. In some embodiments, Xaa1 - Residues, for example, when Xaa1 to Xaa6 are not simultaneously present and one of Xaa7 to Xaa12 represents a glutamic acid residue, the oligopeptide used is tryptophan-tryptophan-glutamic acid. In some embodiments, Xaa1 - Each independently represents any amino acid residue. For example, when Xaa1 to Xaa6 do not exist at the same time, and 2 of Xaa7 to Xaa12 represent aspartic acid residues and arginine residues respectively, the oligopeptide used can be, for example, Tryptophan-tryptophan-aspartic acid-arginine. In some embodiments, Xaa1 - Each independently represents any amino acid residue. For example, when Xaa1 to Xaa6 are not present at the same time, and 3 of Xaa7 to The peptide may be, for example, tryptophan-tryptophan-arginine-glycine-aspartic acid. However, embodiments of the present disclosure are not limited thereto.

In some embodiments, the oligopeptide can be selected from tryptophan-tryptophan, tryptophan-tryptophan-aspartate, tryptophan-tryptophan-arginine-glycine-aspartate , tryptophan-tryptophan-arginine, tryptophan-tryptophan-glutamic acid, tryptophan-tryptophan-glycine, tryptophan-tryptophan-serine, tryptophan-chromotophan Acid-histidine, tryptophan-tryptophan-lysine, tryptophan-tryptophan-tyrosine, tryptophan-tryptophan-cysteine, tryptophan-tryptophan Acid-threonine, preferably, the oligopeptide can be selected from tryptophan-tryptophan, tryptophan-tryptophan-aspartic acid, tryptophan-tryptophan-arginine-glycine-day Partic acid, tryptophan-tryptophan-arginine, tryptophan-tryptophan-glutamic acid. However, embodiments of the present disclosure are not limited thereto.

In some embodiments, the nanocomposition can be tryptophan-tryptophan-cisplatin, tryptophan-tryptophan-aspartate-cisplatin, tryptophan-tryptophan-arginine- Glycine-aspartate-cisplatin, tryptophan-tryptophan-arginine-cisplatin or tryptophan-tryptophan-glutamic acid-cisplatin. In some embodiments, the nanocomposition can be tryptophan-tryptophan-glutamic acid-cisplatin.

In some embodiments, the drug loading of the nanocomposition can be from about 20% to about 60%, such as from about 24% to about 55%, such as from about 24% to about 50%. In some embodiments, the drug loading of the nanocomposition can be 32.1%. In some embodiments, the drug loading of the nanocomposition can be 40.2%. In some embodiments, the drug loading of the nanocomposition can be 30.2%. In some embodiments, the drug loading of the nanocomposition can be 31.2%. In some embodiments, the drug loading of the nanocomposition can be 49.5%.

In some embodiments, the particle size of the nanocomposition may be 10-1000 nm, 20-600 nm, and more preferably 20-200 nm.

pharmaceutical composition

The present disclosure also provides pharmaceutical compositions comprising the above-mentioned nanocomposition and at least one pharmaceutically acceptable excipient.

In some embodiments, pharmaceutically acceptable excipients can be selected from binders, suspending agents, glidants, flavoring agents, disintegrants, dispersants, surfactants, lubricants, commonly used in the art. Colorants, diluents, solubilizers, wetting agents, stabilizers, penetration enhancers, defoaming agents, antioxidants, preservatives, etc. or combinations thereof.

In some embodiments, pharmaceutical compositions can be used to treat tumors.

In some embodiments, the tumor can be lung cancer, breast cancer, gastric cancer, esophageal cancer, adrenocortical cancer, cutaneous squamous cell carcinoma, head and neck cancer, thyroid cancer, liver cancer, pancreatic cancer, bile duct cancer, colorectal cancer, ovarian cancer, Cervical cancer, endometrial cancer, vaginal squamous cell carcinoma, testicular cancer, prostate cancer, bladder cancer, urothelial cancer, melanoma, osteosarcoma, malignant lymphoma, or neuroblastoma. In one embodiment, the tumor may be lung, breast, esophageal, colorectal, or ovarian cancer. In another embodiment, the tumor may be endometrial cancer, vaginal squamous cell carcinoma, testicular cancer, prostate cancer, or bladder cancer. In other embodiments, the tumor may be urothelial carcinoma, melanoma, osteosarcoma, malignant lymphoma, or neuroblastoma.

Typical routes for pharmaceutical compositions of the present application include, but are not limited to, oral, topical, inhalation, parenteral, intranasal, intraocular, intramuscular, subcutaneous, and intravenous administration.

The pharmaceutical composition of the present application can be manufactured by methods well known in the art, such as conventional mixing methods, dissolving methods, granulation methods, sugar-coated pill making methods, grinding methods, emulsification methods, freeze-drying methods, etc.

Preparation

The method for preparing the nanocomposition of the present application is as follows:

Step 1: Dissolve the oligopeptide and cisplatin in the first solvent to obtain organic phase A;

Step 2: Inject the organic phase A obtained in the previous step into the aqueous phase B under stirring; after the addition, continue stirring for 10-60 minutes to obtain mixed solution C;

Step 3: Remove the solvent in mixed solution C to obtain the self-assembled nanocomposition.

In some embodiments, the molar ratio of oligopeptide to cisplatin is about 0.25-20:1, preferably about 0.5-20:1, more preferably about 1-5:1, more preferably about 1-2:1 . In some embodiments, the molar ratio of oligopeptide to cisplatin is about 0.25-2:1. In some embodiments, the molar ratio of oligopeptide to cisplatin is about 1-2:1, and in some embodiments, the molar ratio of oligopeptide to cisplatin is about 1.2-1.8:1. .

In some embodiments, the encapsulation efficiency of the nanocomposition prepared according to the present application can be about 35% to about 99%, such as about 67.5% to about 99%, such as about 80% to about 99%, such as about 86.5% to date 98.2%. In some embodiments, the encapsulation efficiency of the nanocomposition can be 98.2%. In some embodiments, the encapsulation efficiency of the nanocomposition can be 90.7%. In some embodiments, the encapsulation efficiency of the nanocomposition can be 86.5%. In some embodiments, the encapsulation efficiency of the nanocomposition can be 97.3%. In some embodiments, the encapsulation efficiency of the nanocomposition can be 94.3%.

In some embodiments, the drug loading of a nanocomposition prepared according to the present application may be from about 20% to about 60%, such as from about 24% to about 55%, such as from about 24% to about 50%. In some embodiments, the drug loading of the nanocomposition can be 32.1%. In some embodiments, the drug loading of the nanocomposition can be 40.2%. In some embodiments, the drug loading of the nanocomposition can be 30.2%. In some embodiments, the drug loading of the nanocomposition can be 31.2%. In some embodiments, the drug loading of the nanocomposition can be 49.5%.

In some embodiments, the first solvent can be selected from one or more of acetone, ethanol, acetonitrile, tetrahydrofuran, dimethylformamide and dimethyl sulfoxide; preferably, the first solvent is selected from the group consisting of acetone, ethanol and one or more of dimethyl sulfoxide.

In some embodiments, water phase B is selected from one or more of purified water, water for injection, HEPES buffer, Tris buffer or PBS buffer; preferably, water phase B is water for injection or HEPES buffer, More preferably, the pH value of the aqueous phase is 7.0-7.8.

In some embodiments, the organic phase A is injected into the aqueous phase B under stirring conditions, and the volume ratio of the aqueous phase B to the organic phase A can be 1-100:1, preferably 1-40:1.

In some embodiments, the solvent in the mixed liquid C is removed by vacuum evaporation, high-speed centrifugation, dialysis or ultrafiltration, and the vacuum evaporation method is preferably used.

Alternatively, different methods can be used to prepare nanocompositions.

On the other hand, another preparation method of the nanocomposition of the present application is provided, including: dispersing the oligopeptide in the aqueous phase C, then adding cisplatin to obtain a mixed liquid C, and stirring the mixed liquid C at high speed in the dark, The solvent is removed to obtain a colloidal solution of the nanocomposition.

In some embodiments, aqueous phase C can be selected from one or more of purified water, water for injection, HEPES buffer, Tris buffer and PBS buffer. In some embodiments, aqueous phase C is HEPES buffer. In some embodiments, aqueous phase C has a pH of 5 to 9.

In some embodiments, when dispersing the oligopeptide in the aqueous phase C, a dispersion stabilizer may also be added.

In some embodiments, the dispersion stabilizer may be selected from one or more of mPEG2K-NHS, serum albumin, depe-peg2000, lecithin, and plga-peg. In some embodiments, the dispersion stabilizer can be mPEG2K-NHS.

In some embodiments, after adding the dispersion stabilizer, stirring is performed at room temperature for 0.5 to 1.5 hours, preferably 1 hour.

In some embodiments, after dispersing the oligopeptide in the aqueous phase C, a step of dialysis in the aqueous phase D is also included to remove a portion of substances with larger particle sizes.

In some embodiments, aqueous phase D can be selected from one or more of purified water, water for injection, HEPES buffer, Tris buffer and PBS buffer. In some embodiments, aqueous phase D can be water for injection.

In some embodiments, the time of dialysis in aqueous phase D may be 12-36 hours, preferably 18-32 hours, more preferably 24 hours.

In some embodiments, the molar ratio of oligopeptide to cisplatin is about 0.25-20:1, preferably about 0.5-20:1, more preferably about 1-5:1, more preferably about 1-2:1 . In some embodiments, the molar ratio of oligopeptide to cisplatin is about 0.25-2:1. In some embodiments, the molar ratio of oligopeptide to cisplatin is about 1-2:1, and in some embodiments, the molar ratio of oligopeptide to cisplatin is about 1.2-1.8:1.

In some embodiments, the encapsulation efficiency of the nanocomposition prepared according to the present application can be about 35% to about 99%, such as about 67.5% to about 99%, such as about 80% to about 99%, such as about 86.5% to approximately 98.2%. In some embodiments, the encapsulation efficiency of the nanocomposition can be 98.2%. In some embodiments, the encapsulation efficiency of the nanocomposition can be 90.7%. In some embodiments, the encapsulation efficiency of the nanocomposition can be 86.5%. In some embodiments, the encapsulation efficiency of the nanocomposition can be 97.3%. In some embodiments, the encapsulation efficiency of the nanocomposition can be 94.3%.

In some embodiments, the mixture containing the oligopeptide and cisplatin is stirred at high speed at 40-60°C, preferably at 50°C, protected from light.

In some embodiments, the solvent in the mixed liquid C is removed by evaporation under reduced pressure, high-speed centrifugation, dialysis or ultrafiltration, and dialysis is preferably used.

In addition, in some embodiments, the above method may further include the steps of adding a lyoprotectant and freeze-drying to obtain a solid powder.

In some embodiments, the lyoprotectant may be selected from sucrose, trehalose, and mannitol; preferably, the lyoprotectant may be sucrose.

The preparation process of the above-mentioned nanocomposition has mild conditions and simple operation, and the production process does not involve harmful solvents, etc., and is suitable for industrial production.

use

The present disclosure provides the use of the above-mentioned nanocomposition in the preparation of anti-tumor drugs.

The present disclosure also provides the use of the above-mentioned nanocomposition in the preparation of drugs for treating tumors.

Alternatively, the present disclosure provides the above-mentioned nanocomposition for treating tumors.

Alternatively, the present disclosure provides methods of treating tumors, comprising administering to a patient in need thereof The subject is administered a drug containing the above-mentioned nanocomposition, for example, a therapeutically effective amount of a drug containing the above-mentioned nanocomposition.

In some embodiments, tumor refers to cancer.

Non-limiting examples of cancer include, but are not limited to, lung cancer, breast cancer, gastric cancer, esophageal cancer, adrenocortical cancer, cutaneous squamous cell carcinoma, head and neck cancer, thyroid cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, colorectal cancer, ovarian cancer , cervical cancer, endometrial cancer, vaginal squamous cell carcinoma, testicular cancer, prostate cancer, bladder cancer, urothelial cancer, melanoma, osteosarcoma, malignant lymphoma, neuroblastoma treatment drugs. However, embodiments of the present disclosure are not limited thereto.

The dosage forms of the nanocomposition according to the present application include, but are not limited to, ordinary powder, freeze-dried powder, water injection, emulsion, solution and suspension.

In some embodiments, the nanocomposition according to the present application is formed by self-assembly of oligopeptides and drugs driven by π-π stacking, which can achieve efficient loading of drugs; the driving force is physical interaction, which avoids covalent interactions. influence on drug activity.

In some embodiments, the nanocomposition according to the present application has good biocompatibility and safety. The assembly unit tryptophan contained in the oligopeptide is one of the essential amino acids of the human body and participates in the body's protein synthesis and metabolism regulation. Degradation products are endogenous substances in the human body, such as drugs that can significantly reduce the toxic and side effects of tumor treatment.

In some embodiments, the assembly element tryptophan has an isoelectric point of 5.9, triggering its conversion to electropositivity in the acidic environment of endosomes/lysosomes (pH 5.0-5.5), destroying the interaction between the active pharmaceutical ingredient and the oligosaccharide. The force between peptide carriers accelerates the release and accumulation of drugs within the cell.

The following describes the embodiments of the present disclosure with reference to the accompanying drawings. Among them, the experimental methods without specific instructions on the operation steps are all carried out in accordance with the corresponding product instructions. The instruments, reagents, and consumables used in the embodiments can be obtained from Available for purchase by commercial companies.

Abbreviation:

HEPES: 4-Hydroxyethylpiperazineethanesulfonic acid

Tris: Trishydroxymethylaminomethane

DMF: N,N-dimethylformamide

Fmoc-Glu(OtBu)-OH:

Fmoc-Trp(Me)-OH:

WWE:

mPEG2K-NHS: Methoxypolyethylene glycol-active ester

Example 1. Preparation of WWE-cisplatin self-assembled nanocomposition

1.1 Preparation of oligopeptide (WWE)

A. Deprotection

Weigh 1.5g (0.5mmol) of Fmoc-Glu(OtBu)-OH king resin with a substitution degree of 0.34mmol/g, place it in a 100mL peptide synthesis reaction column, add 25mL DMF, mix and soak for 2 hours to fully swell the resin. Add 25mL of deprotecting reagent PIP (20% piperidine/DMF), add nitrogen and mix thoroughly for 45min; wash with DMF (25mL x 6).

Take out a little resin and place it in a 0.5mL EP tube, add 20 μL of 5% ninhydrin absolute ethanol solution, 80 μL of 80% phenol absolute ethanol solution, and heat in a boiling water bath for 5 minutes. If the resin is purple-red, the Fmoc protecting group has been completely removed and the next step of the coupling reaction is carried out; if the resin is colorless or light yellow, the deprotection time needs to be extended.

B. Extended peptide chain

Weigh Fmoc-Trp(Me)-OH (440mg, 1mmol), 1-hydroxybenzotriazole (148mg, 1.2mmol) and 2.5mL DMF, stir to dissolve; add 0.19mL N,N-di Isopropylcarbodiimide (DIC, 1.2mmol), activated for 8 minutes. Add the activated sample to the above-mentioned peptide synthesis reactor and react at room temperature for 1 hour. Use ninhydrin reagent to detect the reaction progress. If the condensation is complete, the resin will be colorless or light yellow; if it is purple-red, you need to extend the condensation time or repeat the condensation.

After the condensation is complete, the resin is washed with DMF (25mL x 6). Follow step A to deprotect Fmoc, and follow step B to proceed to the next condensation of Fmoc-Trp(Me)-OH on the completely deprotected and washed resin.

After the synthesis of the peptide chain, wash it 8 times with absolute ethanol, and then drain the resin.

C. Peptide chain cleavage

Take the above synthetic resin and add 25mL of cleavage reagent (trifluoroacetic acid: anisole thioether: triethylsilane: Ethylene glycol: water = 90:1:2:2:5), stir at room temperature for 2 hours; filter and concentrate; add the concentrated liquid to 10 times the amount of glacial ether to precipitate, centrifuge, add the precipitate to cold ether and repeat The above operation was performed 6 times and dried under reduced pressure to obtain the target peptide.

The structure of the oligopeptide was confirmed by ESI-MS (Figure 1) and ¹ H NMR (Figure 2), [M+Na]+: 570.29.

1.2 Preparation of WWE-cisplatin self-assembled nanocomposites

Weigh tryptophan-tryptophan-glutamic acid (WWE) (4 mg, 7.8 μmol) and disperse it in 2 mL HEPES buffer (pH 8). Add 15 mg mPEG2K-NHS as a dispersion stabilizer, stir at room temperature for 1 hour, and add it to water for injection. Dialyze for 24 hours; add 2 mg of cisplatin (CDDP), stir at high speed for 1 hour in the dark at 50°C, and dialyze to remove free drugs to obtain a colloidal solution.

The appearance of the obtained self-assembled nanocomposition was monodisperse spherical (Figure 3). For ease of description, the WWE-cisplatin self-assembled nanocomposition is called P-DDP. Particle size analysis was performed before P-DDP was lyophilized (Figure 4). Its average particle size was 121 nm and the monodispersity index (PDI) was 0.12. .

1.3 Preparation of pharmaceutical compositions

Add 200 mg of sucrose powder to the colloidal solution obtained in 1.2, and freeze-dry to obtain a solid powder.

Example 2. Influence of preparation conditions

2.1 Effect of feeding ratio on encapsulation efficiency and drug loading capacity

The effect of the ratio of oligopeptide to drug on encapsulation efficiency and drug loading capacity is shown in Table 1 below. For ease of description, the WWE-cisplatin self-assembled nanocomposition is called P-DDP, while free cisplatin is called CDDP, and WWE stands for oligopeptide.

Table 1

Encapsulation rate = (amount of drug in P-DDP/dosing amount) × 100%

Drug loading amount = (amount of drug in P-DDP/(amount of drug in P-DDP + amount of WWE)) × 100%.

Example 3. In vitro release

Weigh 2 mg of P-DDP lyophilized powder and disperse it in 20 mL of HEPES buffer (10 mM, pH 7.4) and 20 mL of PBS (pH 7.4) containing tumor cell lysate. Sample 0.1 mL at specific time points respectively, and centrifuge at 20,000 rpm for 30 min. , draw 20 μL of the supernatant to measure the drug concentration by HPLC, and draw a release curve, as shown in Figure 5.

The P-DDP nanocomposition released slowly in PBS, and the cumulative release of cisplatin lasted for 48 hours; the release of cisplatin from the P-DDP nanocomposition could be accelerated under the conditions of tumor cell homogenate incubation.

Example 4. Serum stability

Disperse 2 mg of P-DDP freeze-dried powder in a buffer (pH=7.4) containing 10% fetal bovine serum (FBS), place it in a 37°C constant-temperature shaker, and shake at 0, 2, 4, 8, and 12 , 24 and 48h were sampled for particle size analysis and compared with the particle size distribution in HEPES buffer (pH8). The results are shown in Figure 6, which shows that there is no significant change in the particle size distribution of P-DDP in 10% FBS within 24h. It shows that the nanocomposition has good stability in blood circulation.

Example 5. In vitro cytotoxicity

As before, according to the CCK8 method, tumor cells in the logarithmic growth phase (human ovarian cancer cell SKOV3 cells and human non-small cell lung cancer cells A549 cells) were seeded into a 96-well plate (5×10 ³ /well), and the cells were allowed to adhere to the wall. Finally, the culture medium was replaced with a culture medium containing P-DDP or CDDP. After continuing to incubate at 37°C for 48 hours, 10 μL of CCK8 reagent was added to each well. After incubation for 4 hours, the OD value of each well was measured at a wavelength of 450 nm, and the cells were calculated. Viability, the results are shown in Figure 7 (SKOV3) and Figure 8 (A549), and the half inhibitory concentration (IC ₅₀ ) of the drug on cell growth was obtained from this, and the results are shown in Table 2.

Table 2

Example 6. In vivo tumor inhibition experiment

150 μL of SKOV3 tumor cell suspension (1×10 ⁷ ) was subcutaneously inoculated into the ventral side of Balb/c nude mice. When the tumor volume reached approximately 150 mm ³ , the tumor-bearing mice were randomly divided into three groups (n=5). , 0.2mL normal saline, 0.2mL 4mg/kg CDDP normal saline solution, 0.2mL 2mg/kg P-DDP normal saline diluent, 0.2mL 4mg/kg P-DDP normal saline diluent and 0.2mL 6mg/ kg P-DDP physiological saline dilution (based on cisplatin content). Each experimental group was administered tail vein administration for 3 consecutive weeks, twice a week. During the administration period, the long and short diameters of the tumors were measured every 2 days, and the tumor volume was calculated. The results are shown in Figure 9. The weight of the tumors was also weighed, and the results were shown in Figure 10.

Example 7. Pharmacokinetics

(1) Establishment of measurement method

① Preparation of platinum standard stock solution (2.5 mg/L): Accurately measure 2.5 mL of platinum standard solution (1 mg/mL), and dilute to 10 mL with 0.2% nitric acid; accurately measure 100 μL, and dilute to 10 mL with 0.2% nitric acid;

②Platinum standard working solution (200μg/L): Accurately measure 0.8mL of platinum standard stock solution, and dilute to 10mL with 0.2% nitric acid;

③Analysis conditions: working wavelength 265.9nm, bandwidth 0.2nm, injection volume 20μL;

④Standard curve: Good linear relationship within the range of 12.5-200μg/L.

(2) Sampling

SD rats (weight 200-250g) were randomly divided into 2 groups (n=5). They were fasted for 12 hours before formal administration and could drink water freely. Each group was given a single injection of 3 mg/kg CDDP physiological saline solution (based on cisplatin content) and 3 mg/kg P-DDP physiological saline dilution into the tail vein at 5 min, 15 min, 30 min, 1 h, 2 h, and 4 h after administration. , 6h, 8h, 12h, 24h, 48h, blood was taken from the orbit and mixed evenly in EP tubes coated with sodium heparin, centrifuged at 5000rpm for 10min to obtain plasma, and stored in a -20°C refrigerator.

Plasma sample processing and determination: Precisely measure 100 μL of plasma sample, add 2 mL of nitric acid-perchloric acid (9:1), place on an electric hot plate and heat until the nitrate is nearly dry. The residue was dissolved with 0.2% nitric acid and the volume was adjusted to 10 mL, measured with a graphite furnace atomic absorption spectrophotometer, and substituted into the standard curve to calculate the blood drug concentration. Taking the blood drug concentration as the ordinate and time as the abscissa, draw the blood drug concentration-time curve, as shown in Figure 11. Kinetica software was used to fit the measured Pt content through a two-compartment model, and various pharmacokinetic parameters were calculated, which are shown in Table 3 below.

table 3

The results showed that the elimination half-life of P-DDP was 18.54h, which was significantly higher than that of the CDDP group (5.62h). The results show that P-DDP can significantly improve the blood circulation time of platinum drugs.

Example 8. Tissue distribution

150 μL of SKOV3 tumor cell suspension (1×10 ⁷ ) was subcutaneously inoculated into the ventral side of Balb/c nude mice. When the tumor volume reached approximately 150 mm ³ , the tumor-bearing mice were randomly divided into 2 groups (n=5). , respectively inject 5 mg/kg CDDP physiological saline solution and 5 mg/kg P-DDP physiological saline dilution (based on cisplatin content) into the tail vein. The mice were sacrificed 12 hours after injection, and different organs such as the heart, liver, spleen, lungs, kidneys and tumors were removed and weighed. Chop the tissue sample into small pieces, add 2 mL of nitric acid-perchloric acid (9:1), and heat on an electric hot plate until the nitric acid solution is almost dry. The residue was dissolved with 0.2% nitric acid and the volume was adjusted to 10 mL. The solution was measured with a graphite furnace atomic absorption spectrophotometer and substituted into the standard curve to calculate the Pt concentration. The results are shown in Figure 12.

The results show that P-DDP has better tumor targeting ability, and the accumulation amount in the tumor site can be as high as 7452ng/g tissue, which is significantly better than the CDDP group (1958ng/g tissue), verifying the targeted delivery of P-DDP efficiency.

Example 9. Safety evaluation

(1) Hemolysis test

Take a few milliliters of rabbit blood, put it into an Erlenmeyer flask containing glass beads and shake it for 10 minutes to remove fibrinogen and turn it into defibrinated blood. Add about 10 times the amount of 0.9% sodium chloride solution, shake well, centrifuge at 1500rpm for 15 minutes, remove the supernatant, and wash the precipitated red blood cells 2-3 times with 0.9% sodium chloride solution according to the above method until the supernatant is no longer visible. until red. The obtained red blood cells were prepared into a 2% (V/V) suspension with 0.9% sodium chloride solution for testing.

Take 7 clean test tubes and number them. Tubes 1-5 are test tubes, tube 6 is negative control tube, and tube 7 is positive control tube. Add 2% red blood cell suspension, 0.9% sodium chloride solution (physiological saline) or distilled water in sequence as shown in Table 4.

Table 4

Each group was set up in 3 parallel groups, vortexed to mix, and then incubated at 37°C for 3 hours. Centrifuge the supernatant and measure the absorbance value at 540nm.

Hemolysis rate % = [(OD sample – OD negative)/(OD positive – OD negative)] × 100%.

The relationship between P-DDP concentration and average hemolysis rate is shown in Table 5 below.

table 5

The results show that the hemolysis rate of P-DDP is <5% in the concentration range of 12.5 to 200 μg/mL.

(2) Weight changes and histopathological examination

Healthy KM mice (5-7 weeks old), weighing 20-23g, were randomly divided into 3 groups (10 mice in each group). Normal saline, 4mg/kg CDDP, 2mg/kg P-DDP, 4mg/kg were injected into the tail vein respectively. kg P-DDP and 6mg/kg P-DDP (calculated as cisplatin content), administered once every 2 days for 5 consecutive times. After administration, regular feeding was performed. The living conditions of the mice were monitored and measured every day. body weight, the results of which are shown in Figure 13. The results showed that the mice in the 4mg/kg CPPD (free cisplatin) group continued to lose weight during the treatment period, and it was observed that eating decreased. The weight of mice in the three concentrations of P-DDP (nanocomposition) groups showed no significant change and the mice were in good condition.

The mice were sacrificed on the 20th day after administration, and the main organs (heart, liver, spleen, lung, and kidney) were collected. After fixing with 4% paraformaldehyde for 24 hours, samples were sent for paraffin embedding, tissue sections were made, and H&E staining was performed. The results of the normal saline group, 4mg/kg CDDP group and 4mg/kg P-DDP group are shown in Figure 14. The results showed that compared with the normal saline group, the liver and kidney sections of mice in the 4 mg/kg CDDP group had obvious lesions; there was no obvious difference in the 4 mg/kg P-DDP group, indicating that the nanocomposition according to the present application did not cause It has obvious organ toxicity, good safety, and avoids the liver and kidney toxicity of free CDDP to a large extent.

Embodiments have been disclosed herein, and although terms are employed, they are used and are to be interpreted in a generic and descriptive sense only and not for purposes of limitation. In some cases, as will be apparent to one of ordinary skill in the art, features, characteristics, and/or elements described with respect to an embodiment may be used alone or in combination with features, characteristics, and/or elements described with respect to other embodiments, unless otherwise Be specific. Accordingly, it will be understood by those of ordinary skill in the art that various changes in form and details may be made without departing from the spirit and scope of the present disclosure as set forth in the claims or their equivalents.

Claims

Nanocomposition, the nanocomposition is formed by self-assembly of an oligopeptide containing a tryptophan dipeptide motif and cisplatin.
The nanocomposition according to claim 1, wherein the general formula of the oligopeptide is Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-W-W-Xaa7-Xaa8-Xaa9-Xaa10-Xaa11-Xaa12,

Where, W is tryptophan; and

Xaa1-Xaa12 each independently does not exist or represents any amino acid residue;
The nanocomposition according to claim 1 or 2, wherein

The oligopeptide is selected from the group consisting of tryptophan-tryptophan, tryptophan-tryptophan-aspartic acid, tryptophan-tryptophan-arginine-glycine-aspartic acid, tryptophan- Tryptophan-arginine, tryptophan-tryptophan-glutamic acid, tryptophan-tryptophan-glycine, tryptophan-tryptophan-serine, tryptophan-tryptophan-histamine Acid, tryptophan-tryptophan-lysine, tryptophan-tryptophan-tyrosine, tryptophan-tryptophan-cysteine, tryptophan-tryptophan-threonine ;

Preferably, the oligopeptide is selected from the group consisting of tryptophan-tryptophan, tryptophan-tryptophan-aspartic acid, tryptophan-tryptophan-arginine-glycine-aspartic acid, tryptophan Acid-tryptophan-arginine, tryptophan-tryptophan-glutamic acid.
The nanocomposition according to any one of claims 1 to 3, wherein

The nanocomposition is tryptophan-tryptophan-cisplatin, tryptophan-tryptophan-aspartic acid-cisplatin, tryptophan-tryptophan-arginine-glycine-aspartate acid-cisplatin, tryptophan-tryptophan-arginine-cisplatin or tryptophan-tryptophan-glutamic acid-cisplatin;

Preferably, the nanocomposition is tryptophan-tryptophan-glutamic acid-cisplatin.
The nanocomposition according to any one of claims 1 to 4, wherein the drug loading capacity of the nanocomposition is about 20% to about 60%, preferably about 24% to about 55%, more preferably about 24% to about 50%.
The nanocomposition according to any one of claims 1 to 5, wherein the particle size of the nanocomposition is 10-1000nm, preferably 20-200nm.
A pharmaceutical composition comprising the nanocomposition according to any one of claims 1 to 6 and at least one pharmaceutically acceptable excipient.
The pharmaceutical composition according to claim 7, the pharmaceutically acceptable excipient is selected from the group consisting of binders, fillers, disintegrants, lubricants, solvents, preservatives, antioxidants, flavoring agents, One or more of fragrances, co-solvents, emulsifiers, solubilizers, osmotic pressure regulators and colorants.
The pharmaceutical composition according to claim 7 or 8, for treating tumors,

Preferably, the tumor is lung cancer, breast cancer, gastric cancer, esophageal cancer, adrenocortical cancer, cutaneous squamous cell carcinoma, head and neck cancer, thyroid cancer, liver cancer, pancreatic cancer, bile duct cancer, colorectal cancer, ovarian cancer, cervical cancer , endometrial cancer, vaginal squamous cell carcinoma, testicular cancer, prostate cancer, bladder cancer, urothelial cancer, melanoma, osteosarcoma, malignant lymphoma or neuroblastoma
The preparation method of the nanocomposition according to any one of claims 1 to 6, including: dispersing the oligopeptide in the aqueous phase C, then adding cisplatin to obtain the mixed liquid C, and stirring the mixed liquid C at high speed in the dark, The solvent is removed to obtain a colloidal solution of the nanocomposition.
The preparation method according to claim 10, wherein

The molar ratio of the oligopeptide to cisplatin is about 0.25-20:1, preferably about 0.5-20:1, more preferably about 1-5:1, more preferably about 1-2:1; and/or

The encapsulation rate of the nanocomposition is about 35% to about 99%, preferably about 67.5% to about 99%, and more preferably about 80% to about 99%.
The preparation method according to claim 10 or 11, wherein

The aqueous phase C is selected from one or more of purified water, water for injection, HEPES buffer, Tris buffer and PBS buffer; preferably, the aqueous phase C is a HEPES buffer; more preferably, the aqueous phase C is a HEPES buffer. The pH of the aqueous phase is 5-9.
The preparation method according to any one of claims 10 to 12, further comprising the step of adding a dispersant,

Preferably, the dispersant is selected from one or more of mPEG2K-NHS, serum albumin, depe-peg2000, lecithin, and plga-peg; preferably, the dispersant is mPEG2K-NHS.
The preparation method according to any one of claims 10 to 13, after dispersing the oligopeptide in the aqueous phase C, further comprising the step of dialysis in the aqueous phase D,

Preferably, the water phase D is selected from one or more of purified water, water for injection, HEPES buffer, Tris buffer and PBS buffer; preferably, the water phase D is water for injection.
The preparation method according to any one of claims 10 to 14, wherein the mixed liquid C is stirred at 40-60°C, preferably at 50°C, in the dark and at high speed.
The preparation method according to any one of claims 10 to 15, wherein the solvent in the mixed liquid C is removed by a reduced pressure evaporation method, a high-speed centrifugation method, a dialysis method or an ultrafiltration method, preferably a dialysis method.
The preparation method according to any one of claims 10 to 16, further comprising the steps of adding a freeze-drying protective agent and freeze-drying to obtain a solid powder,

Preferably, the lyoprotectant is selected from one or more of sucrose, trehalose and mannitol; more preferably, the lyoprotectant is sucrose.
The use of the nanocomposition according to any one of claims 1 to 6 in the preparation of anti-tumor drugs, the anti-tumor drugs being selected from the group consisting of lung cancer, breast cancer, gastric cancer, esophageal cancer, adrenocortical cancer, skin squamous cell carcinoma, Head and neck cancer, thyroid cancer, liver cancer, pancreatic cancer, bile duct cancer, colorectal cancer, ovarian cancer, cervical cancer, endometrial cancer, vaginal squamous cell carcinoma, testicular cancer, prostate cancer, bladder cancer, urothelial cancer , therapeutic drugs for melanoma, osteosarcoma, malignant lymphoma, and neuroblastoma.
The nanocomposition according to any one of claims 1 to 6 or the pharmaceutical composition according to any one of claims 7 to 9 for treating tumors,

Preferably, the tumor is lung cancer, breast cancer, gastric cancer, esophageal cancer, adrenocortical cancer, cutaneous squamous cell carcinoma, head and neck cancer, thyroid cancer, liver cancer, pancreatic cancer, bile duct cancer, colorectal cancer, ovarian cancer, cervical cancer , endometrial cancer, vaginal squamous cell carcinoma, testicular cancer, prostate cancer, bladder cancer, urothelial cancer, melanoma, osteosarcoma, malignant lymphoma or neuroblastoma.
A method of treating tumors, the method comprising administering to a subject in need a drug comprising the nanocomposition of any one of claims 1 to 6 or the pharmaceutical composition of any one of claims 7 to 9,

Preferably, the tumor is lung cancer, breast cancer, gastric cancer, esophageal cancer, adrenocortical cancer, cutaneous squamous cell carcinoma, head and neck cancer, thyroid cancer, liver cancer, pancreatic cancer, bile duct cancer, colorectal cancer, ovarian cancer, cervical cancer , endometrial cancer, vaginal squamous cell carcinoma, testicular cancer, prostate cancer, bladder cancer, urothelial cancer, melanoma, osteosarcoma, malignant lymphoma or neuroblastoma.