阅读说明：本技术 一种靶向乏氧肿瘤的短肽小分子自组装纳米材料及其制备方法和应用 (Short peptide small molecule self-assembly nano material targeting hypoxic tumor, and preparation method and application thereof ) 是由 陈春英 李佳阳 泽纳布·法哈蒂·沙贝特 史可鉴 于 2019-08-23 设计创作，主要内容包括：本发明提供了一种靶向肿瘤乏氧区域的短肽小分子自组装纳米材料的制备方法及其应用,所述纳米材料为短肽修饰的小分子碳酸酐酶抑制剂,其中,所述短肽的N端与取代或未取代的芳香基团结合。通过靶向乏氧肿瘤细胞膜高表达的碳酸酐酶,实现肿瘤微环境原位响应的自组装,并诱发乏氧肿瘤细胞的特异性内吞,从而调控并杀伤乏氧肿瘤细胞。此材料制备工艺简单,具有较高生物安全性,可有效杀伤肿瘤乏氧细胞,显著提高常规临床化疗治疗效果,减缓肿瘤转移进程,为临床肿瘤治疗提供更多思路与手段。(The invention provides a preparation method and application of a short peptide small molecule self-assembly nano material targeting a tumor hypoxia area, wherein the nano material is a small molecule carbonic anhydrase inhibitor modified by a short peptide, and the N end of the short peptide is combined with a substituted or unsubstituted aromatic group. By targeting carbonic anhydrase highly expressed by hypoxic tumor cell membranes, the self-assembly of tumor microenvironment in-situ response is realized, and the specific endocytosis of hypoxic tumor cells is induced, so that the hypoxic tumor cells are regulated, controlled and killed. The material has simple preparation process and higher biological safety, can effectively kill tumor hypoxia cells, remarkably improve the treatment effect of conventional clinical chemotherapy, slow down the tumor metastasis process and provide more ideas and means for clinical tumor treatment.)

1. A short peptide small molecule self-assembly nanometer material for targeting hypoxic tumors is characterized in that the nanometer material is a small molecule carbonic anhydrase inhibitor modified by a short peptide, wherein the N end of the short peptide is combined with a substituted or unsubstituted aromatic group;

preferably, the short peptide consists of 3-5 amino acids; and/or

Preferably, the aromatic group is selected from one or more of the following: benzene ring, pyrene ring, naphthalene ring, fluorenylmethyloxycarbonyl; preferably, the aromatic end capping group is naphthylacetic acid.

2. Nanomaterial according to claim 1, characterized in that the carbonic anhydrase inhibitor is selected from one or more of the following: sulfonamides, sulfamates, dithiocarbamates, xanthates, polyamines, phenol derivatives, coumarin derivatives;

preferably, the carbonic anhydrase inhibitor is 4- (2-aminoethyl) benzenesulfonamide.

3. Nanomaterial according to claim 1 or 2, characterized in that said short peptide comprises a non-natural D-amino acid sequence^DF^DF; and/or

The short peptide comprises a non-natural D-type amino acid sequence^DK；

Most preferably, the short peptide is^DF^DF^DK。

4. The nanomaterial of claim 3, wherein the self-assembled nanomaterial has the following chemical formula:

5. method for the preparation of nanomaterials according to any one of claims 1 to 4, characterized in that it comprises the following steps:

(1) activating the carbonic anhydrase inhibitor;

(2) preparing the short peptide by standard solid phase peptide synthesis methods;

(3) reacting the activated carbonic anhydrase inhibitor obtained in the step (1) with the short peptide obtained in the step (2) at room temperature in a mixed solution of water and methanol to obtain the self-assembly precursor micromolecule of the nano material; and

(4) self-assembling in water solution at room temperature to obtain the nano material;

preferably, in the step (4), the weight percentage of the short peptide small molecule self-assembly nano material in the solution is 0.2 wt% to 1.0 wt%.

6. The method of claim 5, wherein the step (3) further comprises the steps of:

purifying the self-assembly precursor micromolecules obtained in the step (3), and confirming the structure and the purity of the obtained micromolecules;

preferably, the purification method is high performance liquid chromatography.

7. The method according to claim 5 or 6, wherein in the step (3), the nano-materials with different morphologies are obtained by adjusting different pH values;

wherein the self-assembled short peptide small molecule is a clear solution under the condition of pH 9.0; self-assembling the nano-fibers into nano-fibers with the diameter of 10nm to 15nm under the condition of pH 7.0 to 6.5, and forming hydrogel; under the condition of pH5.5-5.0, self-assembling into nano-fiber with the diameter of 200-500 nm.

8. A pharmaceutical composition, comprising:

the nanomaterial of any one of claims 1 to 4; nanomaterial produced by the production method according to any one of claims 5 to 7 and

a pharmaceutically acceptable carrier.

9. Use of a nanomaterial according to any one of claims 1 to 4 or a nanomaterial prepared by the preparation method according to any one of claims 5 to 7 in the preparation of a medicament for the treatment of a tumour.

10. The use of claim 9, wherein the tumor is a hypoxic tumor that specifically expresses high CA IX protease; preferably, the hypoxic tumor is selected from one or more of the following cancers: lung cancer, colon cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, brain glioma, head and neck cancer, oral cancer.

Technical Field

The invention belongs to the field of medicinal chemistry, and particularly relates to a short peptide small molecule self-assembly nano material for targeting hypoxic tumors, and a preparation method and application thereof.

Background

As a common feature in all types of solid tumors, the tumor hypoxia microenvironment has profound clinical significance in multidrug resistance and cancer cell metastasis in tumors. Meanwhile, the tumor hypoxia microenvironment also inhibits immune response and recruits tumor-associated macrophages, thereby protecting the survival of tumor stem cells. During conventional tumor radiotherapy or chemotherapy treatment, the tumor microenvironment can significantly promote cancer cell survival. Alpha-carbonic anhydrases are a large family of 16 distinct phenotypes, of which carbonic anhydrase IX (CA IX) is highly dependent on the tumor hypoxia microenvironment and requires transcriptional activation of hypoxia inducible factor-1 (HIF-1). CA IX, known as a tumor-associated enzyme, is highly overexpressed in hypoxic regions of different tumor types, and confirmed tumor types include cancers associated with brain, neck, lung, bladder, breast, cervix, and the like.

One of the major functions of CA IX in solid tumors is pH regulation: on one hand, CA IX promotes the acidification of the environment at the periphery of tumor cells in a hypoxic microenvironment, which is beneficial to acquiring chemotherapy-resistant and metastasis-resistant phenotypes of hypoxic tumors; on the other hand, this transmembrane metalloenzyme simultaneously helps the intracellular acidic products of hypoxic tumor cells to transport across the membrane, thereby protecting hypoxic cancer cells from acidosis (maintaining a neutral intracellular pH) during the subsequent hypoxia-induced metabolic processes. In addition, current research has confirmed that CA IX enzyme expressed on hypoxic tumor cell membrane can lead tumor cells to carry out endocytosis in hypoxic microenvironment, thereby realizing extracellular substance uptake in hypoxic microenvironment. Given the critical role of the CA IX enzyme in hypoxic tumors, inhibition of the CA IX enzyme action has become an important diagnostic and therapeutic approach in the treatment of hypoxic tumors, e.g., antiproliferation, anti-metastasis, and anti-angiogenesis.

Conventional CA inhibitors, such as sulfonamides and coumarin derivatives, lack sufficient selectivity for tumor-associated CA IX, and they also inhibit other normally functioning CA enzyme subtypes in humans. To enhance their selectivity for transmembrane CA IX enzymes, attempts have been made to modulate the chemical or physical properties of these CA inhibitors. One of the important strategies for selectively targeting CA IX is to decrease its permeability in the cancer cell membrane, allowing the inhibitor to preferentially bind to CA IX enzymes located on the cell membrane surface. Among them, the modification of nanoparticles to improve the selectivity of CA inhibitors has attracted great attention. Since the tunable shape and size of nanoparticles strongly influence the cellular uptake process, CA inhibitor chemically modified nanomaterials are considered as an innovative platform for CA IX targeted therapies.

For example, CA inhibitor modified gold nanorods have achieved more selective uptake into hypoxic cancer cells, resulting in higher efficacy in tumor hyperthermia. Copper oxide nanoparticles carrying CA inhibitors have also been recruited for hypoxic cancer therapy, which selectively targets CA IX and subsequently induces intracellular mitochondrial damage. Although the combination of CA inhibitors and nanoparticles exhibits excellent synergistic effects in hypoxic cancer therapy, current research still faces a series of challenges: for example, the nano material cannot deeply penetrate into a hypoxic region inside a solid tumor, the clinically relevant biological stability and biological safety problems of the nano material, the high preparation cost of the nano medicament in large-scale manufacturing and generation, and the like. How to simply, low-toxicity and effectively enhance the specific targeting of carbonic anhydrase inhibitor small molecules to CA IX enzyme and how to improve the effect of the inhibitor small molecules in the treatment of hypoxic tumors and the related clinical application still become important points of attention of people.

Bio-inspired small molecule self-assembled nanostructures have attracted increasing attention for decades based on natural biocompatibility and biodegradability. Supramolecular nanomaterials mimic the structure and properties of biological systems by the spontaneous assembly of vital elements (e.g. peptides, sugars, lipids, sterols, DNA) through non-covalent interactions. As an innovative biomaterial for biomedical applications, self-assembly of small polypeptide molecules has been widely explored in tissue engineering, immunotherapy, drug-targeted delivery, tumor cell regulation, and tumor cell in/out imaging. Recent studies indicate that the design of polypeptide self-assembly tunable nanostructures triggered by cellular environment is very beneficial for tumor therapy. For example, acidic pH-induced changes in polypeptide nanostructures have been used to stimulate tumor-targeted delivery and subsequent intracellular uptake; two types of polypeptide self-assembly nano structures are provided through differentiated self-assembly activated by extracellular/intracellular environments so as to realize better tumor treatment; the novel strategy of forming the cell surrounding nano-network by inducing polypeptide self-assembly through a tumor microenvironment is used for blocking material exchange among cancer cells, limiting tumor cell migration, helping tumor treatment to target and enrich diagnosis and treatment reagents and the like. Therefore, as an innovative method for modifying the surface of a tumor cell membrane and highly interfering the function of protease related to the cell membrane, the polypeptide supermolecule nanostructure provides an effective strategy for changing the interaction mechanism of the polypeptide supermolecule nanostructure and cells by regulating and controlling the uptake of the tumor cells.

However, self-assembled molecular nanomaterials of short peptides have so far lacked application in hypoxic cancer therapy. Therefore, the present inventors developed a novel nanofiber material of short peptides. The short peptide is used for modifying common CA inhibitor micromolecules to realize the self-assembly of the target hypoxic tumor cell membrane CA IX enzyme and the specific killing of hypoxic tumor cells. The intelligent design can be beneficial to traditional chemotherapy, not only realizes more accurate drug delivery, but also can generate a brand new treatment mechanism, and is a material with high biological safety and simple preparation process. It provides a brand new direction for the treatment of hypoxic tumors and promotes the clinical application prospect of the existing chemotherapy means.

Disclosure of Invention

Therefore, the invention aims to overcome the defects in the prior art and provide a short-peptide small-molecule self-assembly nano material for targeting hypoxic tumors, and a preparation method and application thereof.

Before the technical solution of the present invention is explained, the terms used herein are defined as follows:

the term "CA" means: carbonic anhydrase enzyme.

The term "dd H₂O "means: double distilled water.

In order to achieve the above objects, the first aspect of the present invention provides a short peptide small molecule self-assembled nanomaterial targeting hypoxic tumors, wherein the nanomaterial is a short peptide modified small molecule carbonic anhydrase inhibitor, wherein the N-terminal of the short peptide is bonded to a substituted or unsubstituted aromatic group;

preferably, the short peptide consists of 3-5 amino acids; and/or

Preferably, the aromatic group is selected from one or more of the following: benzene ring, pyrene ring, naphthalene ring, fluorenylmethyloxycarbonyl; preferably, the aromatic end capping group is naphthylacetic acid.

The nanomaterial according to the first aspect of the present invention, wherein the carbonic anhydrase inhibitor is selected from one or more of: sulfonamides, sulfamates, dithiocarbamates, xanthates, polyamines, phenol derivatives, coumarin derivatives;

preferably, the carbonic anhydrase inhibitor is 4- (2-aminoethyl) benzenesulfonamide.

The nanomaterial according to the first aspect of the present invention, wherein the short peptide comprises a non-natural D-form amino acid sequence^DF^DF; and/or

The short peptide comprises a non-natural D-type amino acid sequence^DK；

Most preferably, the short peptide is^DF^DF^DK。

The nanomaterial according to the first aspect of the present invention, wherein the chemical structure of the self-assembled nanomaterial is as follows:

a second aspect of the present invention provides a method for preparing the nanomaterial of the first aspect, the method comprising the steps of:

(1) activating the carbonic anhydrase inhibitor;

(2) preparing the short peptide by standard solid phase peptide synthesis methods; and

(3) reacting the activated carbonic anhydrase inhibitor obtained in the step (1) with the short peptide obtained in the step (2) at room temperature in a mixed solution of water and methanol to obtain the self-assembly precursor micromolecule of the nano material;

and

(4) self-assembling in water solution at room temperature to obtain the nano material;

preferably, in the step (4), the weight percentage of the short peptide small molecule self-assembly nano material in the solution is 0.2 wt% to 1.0 wt%.

The production method according to the second aspect of the present invention, wherein the step (3) further includes the steps of:

purifying the self-assembly precursor micromolecules obtained in the step (3), and confirming the structure and the purity of the obtained micromolecules;

preferably, the purification method is high performance liquid chromatography.

The preparation method according to the second aspect of the present invention, wherein the nanomaterials with different morphologies are obtained by adjusting different pH;

wherein the self-assembled short peptide small molecule is a clear solution under the condition of pH 9.0; self-assembling the nano-fibers into nano-fibers with the diameter of 10-15 nm under the condition of pH 7.0-6.5, and forming hydrogel; under the condition of pH5.5-5.0, self-assembling into nano-fiber with the diameter of 200-500 nm.

A third aspect of the invention provides a pharmaceutical composition comprising:

a nanomaterial as in the first aspect; a nanomaterial produced by the production method according to the second aspect; and

a pharmaceutically acceptable carrier.

In a fourth aspect, the present invention provides the use of the nanomaterial of the first aspect or the nanomaterial prepared according to the preparation method of the second aspect in the preparation of a medicament for the treatment of a tumour.

According to the use of the fourth aspect of the invention, the tumor is a hypoxic tumor specifically highly expressing CA IX protease; preferably, the hypoxic tumor is selected from one or more of the following cancers: lung cancer, colon cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, brain glioma, head and neck cancer, oral cancer.

Traditional CA inhibitors, such as sulfonamides and coumarin derivatives, lack sufficient selectivity for tumor-associated CA IX, while at the same time inhibiting other normally functioning CA enzyme subtypes in humans. Although the combination of CA inhibitors and nanoparticles exhibits excellent synergistic effects in hypoxic cancer therapy, current research still faces a series of challenges: for example, the nano material cannot deeply penetrate into a hypoxic region inside a solid tumor, the clinically relevant biological stability and biological safety problems of the nano material, the high preparation cost of the nano medicament in large-scale manufacturing and generation, and the like. How to simply, low-toxicity and effectively enhance the specific targeting of carbonic anhydrase inhibitor small molecules to CA IX enzyme and how to improve the effect of the inhibitor small molecules in the treatment of hypoxic tumors and the related clinical application still become important points of attention of people.

The invention aims to provide a self-assembly nano material of a targeted hypoxic tumor based on amino acid short peptide with high biocompatibility for modifying common CA inhibitor micromolecules and a preparation method thereof. The inventor simply modifies the common CA inhibitor micromolecule by utilizing the aryl end capping group and the amino acid self-assembly short peptide, so that the self-assembly of the target hypoxic tumor cell membrane CA IX enzyme is realized, and the specific killing of the hypoxic tumor cells is generated. The intelligent design has simple preparation process and high biological safety, can accurately deliver the medicine to the tumor hypoxia area, can realize a brand new treatment mechanism, is beneficial to the application of traditional clinical chemotherapy, and provides a brand new direction for the hypoxia tumor treatment.

The invention utilizes the characteristic of extracellular active sites in CA IX enzyme to enable the small molecules of the CA inhibitor modified by the short peptide to construct the targeting self-assembly of CA IX on the surface of a cell membrane, thereby having the following advantages: (1) these novel CA inhibitor self-assembled nanofibers show greater inhibitory potency against CA IX through greater extracellular retention time and CA IX binding ability; (2) the targeted self-assembly on the hypoxic tumor cell membrane can further block the normal activity of the hypoxic tumor cell, interrupt the regulation and control of CA IX on the pH of the tumor microenvironment and weaken the metastatic migration capacity of the hypoxic tumor cell; (3) the related endocytosis mediated by CA IX promotes the intracellular uptake of the self-assembled nano fibers in a hypoxic microenvironment; (4) the morphology and characteristics of endocytosed nanofibers will also change as the pH in endocytosed vesicles decreases, leading to acidic endocytosed vesicle damage and protective autophagy blockade in hypoxic tumor cells.

The structure-controllable self-assembly nano material with the microenvironment pH response of the hypoxic tumor cell provides a new treatment mechanism and strategy for the hypoxic cancer cell, is beneficial to the clinical application of inhibiting the proliferation and the metastasis of the tumor cell in the treatment of breast cancer tumor, inhibiting the angiogenesis in tumor tissues and the like, can obviously improve the treatment effect of the existing clinical chemotherapy drugs, and provides a brand new direction for the treatment of the hypoxic cancer.

The invention utilizes the characteristic of extracellular active sites in CA IX enzyme to enable short peptide modified CA inhibitor small molecules to construct targeting self-assembly of CA IX on the surface of a cell membrane, and the novel CA inhibitor self-assembly nano fibers show stronger inhibition efficacy on CA IX through stronger extracellular retention time and combination of CA IX capacity. At the same time, this targeted self-assembly on the membrane of hypoxic tumor cells would further block the normal activities of hypoxic tumor cells, including interrupting CA IX regulation of the pH of the tumor microenvironment, and impairing the metastatic migratory capacity of hypoxic tumor cells. More importantly, the related endocytosis mediated by CA IX promotes the intracellular uptake of the self-assembled nanofibers in hypoxic microenvironment, and the shape and characteristics of the nanofibers are changed along with the reduction of the pH value in the endocytosis vesicles in the subsequent endocytosis process, so that the acidic endocytosis vesicle in hypoxic tumor cells is damaged and the protective autophagy is blocked. The structure-controllable self-assembly nano material with the microenvironment pH response of the hypoxic tumor cells provides a new treatment mechanism and strategy for the hypoxic cancer cells, is beneficial to realizing angiogenesis of anti-tumor tissues, proliferation and metastasis of anti-tumor cells and the like, remarkably improves the treatment effect of the existing clinical chemotherapy drugs, and provides a brand new direction for the treatment of the hypoxic cancer.

The specific contents are as follows:

(A) provides a self-assembly nano material design of a targeted hypoxic tumor based on amino acid short peptide modified common carbonic anhydrase inhibitor micromolecules with high biocompatibility, and provides a preparation method of the nano short peptide micromolecules, which comprises the following steps:

(1) synthesizing and preparing self-assembly short peptide: obtaining a short peptide small molecule sequence consisting of 3-5 amino acids by using a classical FMOC solid phase synthesis method, and combining the N-terminal of the short peptide with an aromatic group (such as a benzene ring, a pyrene ring, a naphthalene ring, fluorenylmethyloxycarbonyl and the like) through an amido bond to obtain a short peptide small molecule with good self-assembly capability;

preferably, the self-assembled small short peptide molecule comprises a non-natural D-type amino acid sequence^DF^DF, thereby obtaining the long-acting stable self-assembled nano structure in the organism;

preferably, the self-assembled small short peptide molecule comprises a non-natural D-type amino acid sequence^DK, the side chain amino group of the compound provides a good reaction site for a chemical bond to be combined with a CA inhibitor;

preferably, Naphthaleneacetic Acid (NAP) is an aromatic capping group, attached to an amino acid as an amide, that facilitates the attachment of amino acid short peptides^DF^DF^DK provides better self-assembly effect;

(2) the CA inhibitor micromolecule is used as an important component for targeting hypoxic tumor cells and generating specific inhibition and killing, and the preparation method comprises the following steps: common CA inhibitor small molecules (sulfonamides, sulfamates, dithiocarbamates, xanthates, polyamines, phenol derivatives, coumarin derivatives) are reacted with amino acids via thioureido groups^DThe side chain amino on K is covalently bound;

preferably, the carbonic anhydrase inhibitor small molecule 4- (2-)The primary amino group of aminoethyl) benzenesulfonamide is activated by reaction with N, N' -Thiocarbonyldiimidazole (TCDI) to produce isothiocyanate and obtain stable intermediate product 4- (2-isothiocyanatoethyl) benzenesulfonamide, which is reacted with self-assembled short peptide small molecule NAP-^DF^DF^DK reacts in room temperature water/methanol mixed solution overnight, and is purified by a high performance liquid chromatograph to obtain optimized molecules, wherein the structural formula is as follows:

(B) the invention provides a self-assembly preparation method of the hypoxic tumor targeted short peptide small molecule, which can respond to different pH values inside and outside cells in a hypoxic tumor microenvironment so as to obtain different nanostructures and form nano hydrogel (figure 2).

(C) The material constructs a self-assembly nanofiber targeting CA IX enzyme on the surface of the cell membrane of hypoxic tumor by using the active site of CA IX enzyme outside the cell membrane. This phenomenon was dependent on high expression of CA IX (fig. 3).

(D) The novel carbonic anhydrase inhibitors modified by these amino acid short peptides can enhance the inhibition of CA IX enzyme by virtue of their stronger binding ability to CA IX enzyme protein. One of the most important functions of CA IX enzymes is their pH-regulating ability. Under hypoxic conditions, the CA IX enzyme modulates and lowers the extracellular pH of cancer cells to 6.5, while the intracellular pH will remain around 7.4. This important function not only contributes to the survival of hypoxic cancer cells under glycolytic metabolism, but also to their migratory behavior. Thus, the inventors also demonstrated the enhanced inhibition of CA IX enzyme by the material (fig. 4) by two aspects:

(1) blocking regulation and control of CA IX enzyme on pH of hypoxic tumor microenvironment: the material renders the CA IX enzyme dysfunctional with pH regulation. This alkalization phenomenon in the hypoxic tumor microenvironment can further obstruct anaerobic metabolic pathways in the hypoxic tumor cells, destroying the essential environment on which the hypoxic tumor cells depend for survival.

(2) Reducing metastatic migration ability of hypoxic tumor cells: the oligopeptide modified self-assembly CA inhibitor not only weakens the activity of transmembrane CA IX enzyme, but also limits the normal movement of hypoxic cancer cells, thereby showing stronger effect of inhibiting the metastasis and migration of hypoxic tumors.

(E) On the other hand, the uptake of these self-assembled nanofibers in hypoxic tumor cells is promoted by CA IX enzyme-mediated related endocytosis in the hypoxic tumor microenvironment. The shape and characteristics of the nanofiber are changed along with the reduction of the pH value, and therefore, acidic vesicle damage and protective autophagy blocking in hypoxic cancer cells are caused, and finally, the specific killing of hypoxic tumor cells is caused, and the specific contents are as follows:

(1) the CA IX enzyme-mediated self-assembly nano fibers are subjected to related endocytosis in the hypoxic tumor to cause acid endocytosis vesicle damage of hypoxic tumor cells (figure 5), wherein UHPLC experiments analyze hypoxic tumor cell membranes and tumor cell lysates treated by the material, and the targeted self-assembly and specific endocytosis of the material to the hypoxic tumor cells are confirmed (figure 6);

(2) according to the invention, the endocytosis of the CA IX enzyme-mediated self-assembled nanofiber is verified by monitoring the relevant signal pathway of tumor cell protective autophagy under hypoxic conditions, so that the normal function of protective autophagy in hypoxic tumor cells is further blocked (fig. 7).

(3) The invention proves that the short peptide self-assembly small molecule nano material can generate specific killing on hypoxic tumor cells through cytotoxicity experiments, the killing effect on the tumor cells in a hypoxic environment is obviously higher than that of normal oxygen, and the treatment effect is obviously better than that of other control groups, including an unmodified commercial CA inhibitor small molecule control group and a short peptide control group which is not connected with a CA inhibitor (figure 8).

(F) The invention also aims to provide a new treatment mechanism and strategy for the hypoxic tumor cells by using the structure-controllable self-assembly nano material with the microenvironment pH response, which is beneficial to realizing the benefits of inhibiting the proliferation and the metastasis of the tumor cells, inhibiting the angiogenesis in tumor tissues and the like in the treatment of breast cancer and remarkably improving the treatment effect of the conventional clinical chemotherapy drugs. (can be expanded to the brain, neck, lung, bladder, breast, cervix and the like with tumor hypoxia microenvironment CA IX overexpression)

(1) So far, in vitro cell experiments have revealed the great potential of the material for selectively inhibiting hypoxic MDA-MB-231 tumor cells, and animal experiments carried out by the inventor prove that the treatment effect of the design for inhibiting hypoxic microenvironment in living tumors is:

animal experiments using a nude mouse model carrying breast cancer xenografts only the material-treated group significantly attenuated the relevant biomarker signals (HIF-1. alpha. and CA IX) for hypoxic tumors, meaning that the degree of hypoxia in solid tumors was successfully attenuated (see FIG. 9). Notably, none of the remaining control treatments showed significant efficacy in inhibiting hypoxia. In addition, a unique increase in autophagosome accumulation (e.g., punctate structure of LC3B) and a clear attenuation in cancer cell proliferation (Ki67 fluorescence decrease) were seen in the material treatment group, again validating the in vitro cell assay results. Thus, the material does have a comparable therapeutic effect on hypoxic tumors in vivo.

(2) Due to the key role of CA IX enzymes in the hypoxic tumor microenvironment, CA IX inhibitors can inhibit tumor metastasis and angiogenesis in tumor therapy. The inventor has shown some relevant effects in previous in vitro cell experiments, and therefore, the inventor further verifies the potential application of the inventive material in inhibiting metastasis and angiogenesis through animal experiments. The present inventors used a homologous mouse metastasis model of murine 4T1 breast cancer cells, statistically analyzed the number of metastases in lung tissue, and directly confirmed that the material has a significant tumor metastasis inhibition function (fig. 10). After detecting the vascular endothelial marker CD31 in tumor tissues, the inventors further found that in the material treatment group, the tumor blood vessel-associated CD31 immunofluorescence image becomes broken and weak, while the tumor blood vessels in the control group maintain relatively intact structures, which suggests that the material of the invention has the efficacy of inhibiting tumor angiogenesis in hypoxic tumor tissues.

(3) By inhibiting the related biological functions of CA IX, the clinical application and curative effect of conventional chemotherapy are effectively promoted. Hypoxic microenvironments have been identified as key factors for clinical tumor growth, metastatic spread and multidrug resistance. CA IX is specifically overexpressed in the tumor hypoxic environment of a variety of cancers, regulates the pH of the tumor microenvironment, and helps solid tumors possess highly metastatic and anti-chemotherapeutic properties. To date, the present inventors have demonstrated that the material will effectively disrupt the hypoxic microenvironment within solid tumors by inhibiting CA IX, thereby affecting tumor proliferation, metastasis and angiogenesis. Here, the inventor further proves through animal experiments that the material can promote the use of the conventional clinical chemotherapy drugs and enhance the anti-tumor effect of the conventional clinical chemotherapy drugs under the condition of low-dose administration.

The present inventors have developed a combination therapy of the material with conventional clinical chemotherapeutic drugs, preferably using doxorubicin (Dox) at low doses.

The present inventors established a tumor xenograft nude mouse model, and the combined use of the material and low dose Dox effectively achieved a reduction in tumor volume (fig. 11) and was significantly superior to the Dox group administered alone. This indicates that the material treatment can effectively enhance the sensitivity of tumor to Dox, and the Dox treatment can show significant anti-tumor performance even at relatively low dosage by destroying the hypoxic microenvironment of solid tumor, thereby accelerating the effect of conventional chemotherapy treatment.

(4) In addition, the safety of the material in animal experiments is also evaluated.

Preferably, the inventors monitored the toxic effects of the nanomaterial-treated group. The material treatment group has no obvious abnormality on the body weight and organ structure of the mouse, and the biocompatibility of the material treatment group in-vivo application is ensured;

preferably, the inventors monitored the toxic effects of the combination treatment group. The combination treatment did not die throughout the treatment period. The combined use of the material and Dox did not significantly reduce the body weight of the mice when compared to the control group. Biochemical indicators of blood (e.g., alanine transaminase, aspartate transaminase, and creatinine) further reflect the lack of significant severe liver and kidney damage following combination treatment of the material with Dox (fig. 12).

Along with the rapid proliferation of tumors, hypoxic microenvironments have profound clinical significance on tumor growth and metastasis, which not only significantly increase the difficulty of drug diffusion in tumor sites, but also protect tumor stem cells and significantly improve the survival capability of tumor cells during traditional tumor radiotherapy or chemotherapy. However, the application of the nano-material in the treatment of hypoxic tumors is limited by the problems of biological safety, permeability, material stability and the like. As a hypoxia-induced transmembrane enzyme, CA IX is a specific therapeutic diagnostic target for hypoxic tumors. By utilizing the extracellular active site in the CA IX enzyme, the inventor modifies the CA inhibitor by D-type amino acid, and therefore, develops the targeted hypoxic self-assembly nanofiber constructed by the short peptide. The CA IX targeted self-assembly is successfully realized on the cell membrane surface of the hypoxic tumor, and the nanofiber consisting of the novel CA inhibitors shows stronger inhibition efficacy on the CA IX based on longer extracellular retention time and higher binding capacity. In addition, tumor cells in hypoxic environments mediate endocytosis of these nanofiber materials by CA IX enzymes and cause hypoxic tumor cell-specific organelle damage and protective autophagy blockade.

These interesting in vitro phenomena and mechanisms of action motivate the present inventors to further explore the potential applications of the hypoxic-targeted self-assembled nanomaterials in vivo. First, their ability to inhibit hypoxic microenvironments, such as immunofluorescence imaging, flow cytometry and Western blotting, was studied by various methods and tools in vivo. In support of these results, the present inventors designed this novel self-assembling CA inhibitor to achieve significant therapeutic effects in hypoxic tumors by inhibiting CA IX. Thereafter, the inventors have verified in the 4T1 breast cancer model that the material significantly inhibits CA IX-associated tumor metastasis and angiogenesis. More interestingly, as a means of conventional clinical chemotherapy, Dox treatment has made a significant advance with the aid of the material. Losing the protection of the hypoxic microenvironment, solid tumors appear to be more sensitive and intolerant to Dox administration. As the present inventors know, Dox is still currently challenged with various side effects such as cardiotoxicity, allergic reactions, abnormal pigmentation, treatment-related leukemia, etc. Long-term chemotherapy can be dangerous and painful for the patient, while also increasing the risk of developing drug resistance. Therefore, cancer intervention treatment is receiving increasing attention because of its excellent effects in adjuvant treatment and pain relief. It provides local treatment (e.g. intravascular tamponade and local ablation) and has recently been applied to the treatment of cancers such as esophageal cancer, colorectal cancer, lung cancer and primary liver cancer. In view of the critical role of CA IX enzyme in clinical settings, the administration of the novel self-assembling CA inhibitor nanomaterials to tumor hypoxic regions can also be a superior choice for cancer intervention. It not only inhibits CA IX-associated tumor angiogenesis, but is also effective in inhibiting hypoxic regions where there is no vascular transport of oxygen and nutrients. Thanks to its biocompatible components and superior hypoxic therapeutic effect, the novel self-assembled CA inhibitor can be a safe and convenient material, promoting current clinical chemotherapy approaches.

Finally, it is also important that the target-specific nanostructure is constructed by self-assembly of short peptide modified anticancer drugs, providing the inventors with more therapeutic tool options. The huge investment, long cycle and high risk challenges make innovative development of small molecule drugs enter the bottleneck. However, in recent years, new use of old drugs has become a new direction for drug development. Different from the development of common drugs, the research and development strategy can shorten the clinical test time, reduce the cost, improve the success rate and effectively solve various key problems of the traditional innovative drug development. It has recently been found that many conventional drugs, such as anti-inflammatory drugs aspirin, alcohol withdrawal drug disulfiram, diabetes drug metformin, etc., have a completely new mechanism and exhibit unexpected biological effects in anticancer therapy. Inspired by the strategy, the inventor provides a new idea for modifying the traditional medicine by using a plurality of amino acids and exploring a brand new action mechanism of the traditional medicine by using the new functions and the self-assembled nano structure of the traditional CA inhibitor.

The nanomaterial of the present invention may have the following beneficial effects, but is not limited to:

(1) the invention utilizes amino acid short peptide with high biocompatibility to modify common carbonic anhydrase inhibitor micromolecule, and the novel self-assembled carbonic anhydrase inhibitor nano material is a material with high biological safety and simple preparation process.

(2) The nano material can realize the self-assembly design of the target hypoxic tumor cell membrane CA IX enzyme, and generate nano fiber structures with different sizes by responding different pH conditions outside and inside the hypoxic tumor microenvironment, thereby showing the specific killing of the hypoxic tumor cells.

(3) The hydrogel nano material of the self-assembled carbonic anhydrase inhibitor obviously enhances the anti-tumor efficacy of the traditional chemotherapeutic drugs. Due to the good biocompatibility and excellent hypoxic tumor curative effect, the novel short peptide modified self-assembled carbonic anhydrase inhibitor can be a safe and convenient material, promotes the clinical application development of the existing chemotherapy means, and provides a brand new direction for hypoxic cancer treatment.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

fig. 1(a) shows nuclear magnetic data of targeted hypoxic self-assembled short peptide small molecules according to the invention; fig. 1(b) shows mass spectrum data of the targeted hypoxic self-assembled short peptide small molecule of the invention.

Fig. 2(a) shows the macro morphology and the electron microscope morphology of the nano-material at different pH, and fig. 2(b) shows the rheological data of the nano-hydrogel at different pH.

FIG. 3(a) shows a photograph of a hypoxic tumor cell culture dish; environmental scanning electron microscope: the short peptide self-assembly micromolecule targets the surface of the cell membrane of the hypoxic tumor to form a reticular fiber structure; FIG. 3(b) shows the CA IX mRNA and protein levels in hypoxic tumor cells.

Fig. 4 shows the change in color of the hypoxic tumor cell culture medium after treatment with the material, fig. 4(a), and fig. 4(b) shows the metastatic migratory capacity behaviour of the hypoxic tumor cells.

Fig. 5 shows the results after treatment of the material (N-pepABS (+) panel), where fig. 5(a) shows significant numbers of CA IX and endocytic vesicle fluorescent co-localization of hypoxic tumor cells, followed by fig. 5(B) shows acidic vesicle swelling of hypoxic tumor cells, followed by fig. 5(C) shows fluorescent staining confirming acidic vesicle damage within hypoxic tumor cells, and fig. 5(D) electron microscope images showing a large number of nanofibrous structures observed within the cytoplasm of hypoxic cells.

Figure 6 shows the change in the amount of intracellular enrichment and hypoxia on the membrane of hypoxic tumor cells of the material (N-pepABS (+) group) following knock-down of CA IX protein expression, thus demonstrating that both hypoxia-targeted self-assembly and specific endocytosis of the material are CA IX dependent: fig. 6(a) shows the reduction of CA IX expression in hypoxic tumor cells by gene interference experiments, followed by fig. 6(B) shows flow results demonstrating that the degree of acidic vesicle damage caused by the material is positively correlated with CA IX expression, followed by UHPLC results demonstrating that fig. 6(C) shows that the nanomaterial concentration enriched on the membrane of hypoxic tumor cells also has CAIX dependence, and fig. 6(D) shows that the nanomaterial concentration enriched in the cytosol (cell lysate) of hypoxic tumor cells also has CA IX dependence.

Figure 7 shows that acid vesicle damage blocks protective autophagy in MDA-MB-231 cells: FIG. 7(A) shows the mRNA level ratio of Atg5/GADPH 48 hours after the cells were treated with the material; FIG. 7(B) shows a fluorescence image of autophagosome accumulation in the cytoplasm of MDA-MB-231 cells; FIG. 7(C) shows a Western blot analysis of autophagy-related signals; FIG. 7(D) shows an autophagy flow study in MDA-MB-231 cells, pretreated for 10nM Baf 1 hours, and then the material was treated for 48 hours.

Figure 8 shows the results of the cellular activity of tumor cells under hypoxic/normoxic conditions (CCK-8 experiment) 72 hours after treatment of the material.

Fig. 9 shows immunofluorescence images of hypoxic tumor-associated biomarkers (a) CA IX and (B) HIF-1A, respectively, exhibited in solid tumors, and fig. 9(C) and 9(D) show immunofluorescence images of (C) autophagy-associated protein LC3B and (D) tumor cell proliferation-associated Ki67 within tumor tissues, in animal in vivo experiments.

Fig. 10 shows in tumor metastasis models, fig. 10(a) shows that the material-treated group (N-pepABS) significantly reduced the number of metastases appearing in lung lobe tissues of mice, and fig. 10(B) shows that tumor metastasis-inhibiting effects with statistically selected differences were exhibited compared to the solvent-control group (Con); after the material treatment, fig. 10(C) and 10(D) show that both (C) hypoxic-associated CA IX signal and (D) tumor vascular tissue-associated biomarker CD31 within tumor tissue show significant differences.

Fig. 11 shows that after the material (N-pepABS) was used in combination with low dose doxorubicin (Dox), fig. 11(a) shows the volume and fig. 11(C) shows the weight of significantly inhibited tumor growth, and fig. 11(B) shows that a statistically significant difference was exhibited compared to the solvent control group (Con); after the material was used in combination with low dose Dox, fig. 11(D) shows the most significant decrease in CA IX enzyme expression in tumor tissue, fig. 11(E) shows the most significant formation of necrotic regions in tumor tissue, fig. 11(F) shows the most significant decrease in cell proliferative activity in tumor tissue, and fig. 11(G) shows the most significant destruction of tumor vasculature in tumor tissue.

Fig. 12(a) shows the evaluation of the biosafety of mice after the material treatment, including no abnormality in body weight and various organ mechanisms; figure 12(B) shows the evaluation of the biological safety of mice after the material was treated in combination with low dose Dox, including no significant abnormalities in body weight, liver function and kidney function.

Detailed Description

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

This section generally describes the materials used in the testing of the present invention, as well as the testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well within the skill of the art, provided that they are not specifically illustrated.

The reagents and instrumentation used in the following examples are as follows:

reagent:

4- (2-aminoethyl) benzenesulfonamide from BIOMOL, DMF from ALFA, 1,1' -thiocarbonyldiimidazole, methanol and anhydrous dichloromethane from Macklin, ethyl acetate from Acros, N-hexane from Aldrich, 2-chlorotrityl chloride resin from Aisabio, Fmoc-D-Lys-Boc-OH from SIGMA, N, N-diisopropylethylamine from MERYER, piperidine from Acmec, triethylamine and N ', N ' -tetramethyl-hexafluorophosphate from TCI, DCM from GE, concentrated hydrochloric acid from Amresco, PBS from WISENT, crystal violet from TargetMol, doxorubicin from RHAWN, MDA-MB-231 and Hela cells, etc. from ATCC, culture medium from Invitrogen, immunofluorescent antibodies of various types from Abcam, Cell Signaling, technology, System Biosciences, Earth ox;

the instrument comprises the following steps:

transmission electron microscope, model Morgagni 268 Transmission Electron microscope.

Magnetic resonance imaging apparatus, model number Varian Unity Inova 400

Flow cytometer available from Bidi medical instruments Inc. model BD Accuri C6

Confocal microscope, model Zeiss 710 from Zeiss optical group, Germany

HPLC from Waters under the model Waters Delta600HPLC system

High performance liquid chromatography semi-preparative column available from Waters under the XTerra C18 RP column

High performance liquid chromatograph available from Shimadzu corporation, model LC20 AT.