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CN116139293B - An amphiphilic molecule for targeting and regulating tumor angiogenesis and an acid-responsive peptide nanodrug targeting tumor tissue - Google Patents

An amphiphilic molecule for targeting and regulating tumor angiogenesis and an acid-responsive peptide nanodrug targeting tumor tissue Download PDF

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CN116139293B
CN116139293B CN202210826228.0A CN202210826228A CN116139293B CN 116139293 B CN116139293 B CN 116139293B CN 202210826228 A CN202210826228 A CN 202210826228A CN 116139293 B CN116139293 B CN 116139293B
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CN116139293A (en
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黄涛
孙献甫
王倩
葛红
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Henan Cancer Hospital
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Abstract

本发明提供了一种靶向调控肿瘤血管新生的两亲性分子，属于功能型多肽纳米技术领域，所述两亲性分子包括亲水端和疏水端，所述亲水端为抗血管新生多肽，所述抗血管新生多肽的N端与6‑氨基己酸通过酰胺键相连，所述6‑氨基己酸另一端连接所述疏水端，所述疏水端为3‑二乙氨基丙基异硫氰酸酯。所述两亲性分子能够作为药物载体，延长抗肿瘤药物半衰期，且具有肿瘤部位特异性响应，在肿瘤弱酸性微环境下响应释放药物，使药物高效运输至肿瘤部位，所述两亲性分子还能够靶向抑制肿瘤血管新生，协同提升抗肿瘤药物的疗效。本发明还提供了一种靶向肿瘤组织的酸响应型多肽纳米药物。

The present invention provides an amphiphilic molecule for targeted regulation of tumor angiogenesis, belonging to the field of functional polypeptide nanotechnology, wherein the amphiphilic molecule includes a hydrophilic end and a hydrophobic end, wherein the hydrophilic end is an anti-angiogenic polypeptide, wherein the N-terminus of the anti-angiogenic polypeptide is connected to 6-aminocaproic acid by an amide bond, and the other end of the 6-aminocaproic acid is connected to the hydrophobic end, wherein the hydrophobic end is 3-diethylaminopropyl isothiocyanate. The amphiphilic molecule can be used as a drug carrier to extend the half-life of anti-tumor drugs, and has a tumor site-specific response, responds to release drugs under the weakly acidic microenvironment of the tumor, and enables the drug to be efficiently transported to the tumor site, and the amphiphilic molecule can also target and inhibit tumor angiogenesis, and synergistically improve the efficacy of anti-tumor drugs. The present invention also provides an acid-responsive polypeptide nanomedicine targeting tumor tissue.

Description

Amphiphilic molecule for targeted regulation of tumor angiogenesis and acid response type polypeptide nano-drug for targeted tumor tissue
Technical Field
The invention belongs to the technical field of functional polypeptide nanometer, and in particular relates to an amphiphilic molecule for targeting and regulating tumor angiogenesis and an acid response type polypeptide nanometer drug for targeting tumor tissues.
Background
According to the report of 2020 world cancer issued by the world health organization, cancer has become a main killer threatening human health, and mortality and morbidity are still continuously increasing. Uncontrolled proliferation, invasiveness to surrounding tissue cells, and metastatic nature in other parts of the body are important features of malignant tumors. The most important treatment strategies aiming at malignant tumors in clinic are surgical excision and radiotherapy and chemotherapy treatment, and the most important problems faced by the current tumor treatment are still that medicines are difficult to transport to tumor sites and relapse is easy to occur after treatment. Among the chemotherapy drugs, the polypeptide drug has unique advantages that the drug has higher drug resistance than general chemical drugs, high bioactivity, strong specificity and relatively weak toxic reaction, and is not easy to accumulate in vivo. However, compared with small molecule chemicals, the polypeptide molecules have the disadvantages of poor stability and easy degradation in vivo, thus having short half-life, requiring continuous administration to maintain the drug effect, and causing inconvenience to patients.
Polypeptide nanomaterials are widely used for in vivo transport of drugs due to their passive targeting properties. The polypeptide nanoparticle has good biocompatibility, high safety, strong designability and certain targeting property, and can protect the polypeptide drug from contacting protease, thereby prolonging the half-life period of the polypeptide drug. Polypeptide self-assembly refers to a molecular aggregate or supermolecular structure which is formed by spontaneous combination of amino acid residues through non-covalent bond interaction under proper conditions, has definite structure and stable structure and has certain physical and chemical properties. Non-covalent interactions are critical for self-assembly of molecules, and common non-covalent interactions include hydrogen bonding, van der Waals forces, electrostatic interactions, hydrophobic interactions, pi-pi stacking interactions, cationic adsorption interactions, and the like. The non-covalent interactions maintain the structural stability and integrity of the self-assembled system.
Disclosure of Invention
In order to solve the problem of poor curative effect of anti-tumor drugs, the invention provides an amphiphilic molecule for targeted regulation of tumor angiogenesis, which can be used as a drug carrier, prolong the half life of the anti-tumor drugs, has tumor site specific response, responds to release drugs in a slightly acidic microenvironment of the tumor, enables the drugs to be transported to the tumor site efficiently, can also targeted inhibit tumor angiogenesis, and synergistically improves the curative effect of the anti-tumor drugs.
The invention also provides an acid response type polypeptide nano-drug for targeting tumor tissues.
The invention is realized by the following technical scheme:
an amphiphilic molecule for targeted regulation of tumor angiogenesis, wherein the amphiphilic molecule comprises a hydrophilic end and a hydrophobic end, the hydrophilic end comprises an anti-angiogenesis polypeptide and a connecting peptide, the N end of the anti-angiogenesis polypeptide is connected with the connecting peptide through a peptide bond, the N end of the connecting peptide is connected with 6-aminocaproic acid through an amide bond, the other end of the 6-aminocaproic acid (Acp) is connected with the hydrophobic end through a thiourea bond, and the hydrophobic end is 3- (diethylamino) propyl isothiocyanate.
Further, the amino acid sequence of the hydrophilic end is shown in SEQ ID NO. 1.
The amino acid sequence of the anti-angiogenesis polypeptide is PCAIWF, the amino acid sequence of the connecting peptide is DDEE, and the amino acid sequence of the hydrophilic end is DDEEPCAIWF.
Based on the same inventive concept, the application also provides application of the amphiphilic molecule for targeted regulation of tumor angiogenesis in preparation of a drug carrier or preparation of a targeted regulation of tumor angiogenesis drug.
Based on the same inventive concept, the application also provides an acid response type polypeptide nano-drug targeting tumor tissues, wherein the polypeptide nano-drug comprises a hydrophobic anti-tumor drug and a drug carrier coated outside the hydrophobic anti-tumor drug, and the drug carrier is an amphiphilic molecule for targeting and controlling tumor angiogenesis.
Optionally, the hydrophobic anti-tumor drug comprises a hypoxia prodrug.
In the application, the medicine coated by the medicine carrier is not limited to the hypoxia prodrug, but can be other hydrophobic small molecule medicines or other hydrophobic antitumor medicines.
Optionally, the hydrophobic antitumor drug comprises any one of AQ4N (barnoquinone), TPZ (Ti rapazamine/tirapazamine), TH-302 (Evofosfamide) and Car icotamide.
Further, the particle size of the polypeptide nano-drug is 25-30nm, and the average particle size is 26.52nm.
Based on the same inventive concept, the application also provides a preparation method of the acid-responsive polypeptide nano-drug targeting tumor tissues, which comprises the following steps:
Co-dissolving amphiphilic molecules and hydrophobic antitumor drugs in an organic phase;
dispersing an organic phase dissolved with amphiphilic molecules and hydrophobic antitumor drugs in a water phase under an ultrasonic condition to obtain the polypeptide nano-drug.
Further, the organic phase comprises dimethyl sulfoxide and the aqueous phase comprises deionized water or phosphate buffer at pH 7.4.
Further, dispersing an organic phase dissolved with amphiphilic molecules and hydrophobic antitumor drugs in a water phase under the ultrasonic condition to obtain the polypeptide nano-drugs, which specifically comprises the following steps:
Dispersing the organic phase dissolved with amphiphilic molecules and hydrophobic antitumor drugs in the water phase under the action of ultrasonic waves with power of 80-120W, and standing for 1-4h for 10-30min to obtain the polypeptide nano-drugs.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
1. The amphiphilic molecule for targeted regulation of tumor angiogenesis has the hydrophilic end of anti-angiogenesis polypeptide and connecting peptide, the hydrophobic end of 3-diethylaminopropyl isothiocyanate, and the hydrophilic end and the hydrophobic end of the amphiphilic molecule are connected through (Acp), the amphiphilic molecule can be used as a drug carrier to prolong the half life of an anti-tumor drug, has tumor site specific response, and can be depolymerized in a slightly acidic environment of the tumor due to the protonation of the 3- (diethylamino) propyl isothiocyanate, so that the anti-tumor drug molecule is specifically released to enable the drug molecule to be transported to the tumor site with high efficiency.
2. The amphiphilic molecule is depolymerized in response to an acidic environment, an anti-tumor drug molecule is released, and simultaneously, an anti-angiogenesis polypeptide is specifically released, wherein the anti-angiogenesis polypeptide has the capability of targeting Vascular Endothelial Growth Factor Receptor (VEGFR) in tumor tissues, antagonizes the VEGFR after targeting, thereby regulating and controlling the tumor-related angiogenesis, and can also target and inhibit tumor angiogenesis, so that the anti-angiogenesis polypeptide plays a role in combining with the drug molecule.
3. According to the amphiphilic molecule for targeted regulation of tumor angiogenesis, a hydrophilic end and a hydrophobic end of the amphiphilic molecule are connected through (Acp), 6-aminocaproic acid is also called an alkyl spacer, 3- (diethylamino) propyl isothiocyanate can react with-SH and side chain-NH 2 in a polypeptide structure in an amphiphilic molecule synthesis reaction, the possibility of side reaction is reduced due to the addition of Acp, six-carbon linear space is provided by Acp, the steric hindrance of the reaction is obviously reduced, the reaction efficiency is improved, the reaction difficulty is reduced, in addition, when the anti-angiogenesis polypeptide responds under the acidic environment condition of a tumor, the situation that the last amino acid is usually cut off is avoided due to the access of the alkyl spacer Acp, so that the released polypeptide cannot effectively inhibit the angiogenesis of the tumor, on one hand, the connecting peptide plays a role in connection, and on the other hand, the hydrophilicity of the hydrophilic end can be increased.
4. The invention relates to an acid response type polypeptide nano-drug for targeting tumor tissues, which comprises a hydrophobic anti-tumor drug and a drug carrier coated outside, namely an amphiphilic molecule, wherein compared with the single anti-tumor drug molecule, the half life of the polypeptide nano-drug is longer, the anti-tumor drug molecule and the anti-angiogenesis polypeptide can be targeted and released in a slightly acidic environment of the tumor, the anti-angiogenesis polypeptide can inhibit angiogenesis of the tumor sites while the anti-tumor drug molecule is efficiently transported to the tumor sites, so that the tumor tissues are gradually reduced due to lack of oxygen and nutritional components, and the synergistic anti-tumor effect with the hydrophobic anti-tumor drug is realized.
5. The invention relates to an acid response type polypeptide nano-drug for targeting tumor tissues, wherein hydrophobic antitumor drugs can adopt hypoxia prodrugs such as AQ4N, the hypoxia prodrugs are nontoxic and generate active drugs through reductase catalysis under the condition of hypoxia, the activity of topoisomerase II in tumor cells is inhibited, and then the tumor cells are killed, the anti-angiogenesis polypeptide released by tumor response further aggravates the hypoxia condition in the tumor microenvironment by inhibiting angiogenesis, and obviously improves the capability of the hypoxia prodrug to kill tumor cells.
6. The acid response type polypeptide nano-drug targeting tumor tissues is formed by co-assembling amphiphilic molecules and hydrophobic anti-tumor drugs under an ultrasonic condition to form a highly ordered nano-structure, the particle size of the polypeptide nano-drug is 26.52nm on average, the particle size is small, the polypeptide nano-drug is favorable for endocytosis of tumor cells, has in vivo stability, is not easy to degrade, does not generate covalent bonds in the self-assembly process, has no adverse reaction, and the product has the characteristics of good biocompatibility, high safety and the like, and is expected to become a good alternative biomedical material.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a biological transmission electron microscope image of the prepared polypeptide nano-drug under physiological conditions.
FIG. 2 shows the biological transmission electron microscope images of the prepared polypeptide nano-drug under the conditions of pH 7.4 and pH 6.8.
FIG. 3 shows the migration ability of HUVEC cells with the prepared polypeptide nano-drug, and the image (part A) and the statistical analysis (part B) thereof.
FIG. 4 is a diagram showing a potential mechanism of inhibiting HUVEC cell migration by the prepared polypeptide nano-drug, wherein A represents the expression of protein immunoblotting detection proteins ERK1/2 and p-ERK1/2, and B represents the quantitative result of Image J software on panel A.
FIG. 5 shows the effect of the prepared polypeptide nano-drug on the activity of tumor cells in the state of hypoxia and oxygen enrichment.
FIG. 6 shows the effect of the prepared polypeptide nano-drug on promoting tumor cell apoptosis in the state of hypoxia and oxygen enrichment, wherein A shows the effect of different treatments on cell apoptosis detected by a flow cytometer, and B shows the statistical result on the apoptosis proportion of the A graph.
FIG. 7 shows potential mechanism analysis of the prepared polypeptide nano-drug for promoting tumor cell apoptosis in the state of hypoxia and oxygen enrichment, wherein, A is a graph showing that Western immunoblotting Western Blot detects hypoxia index HIF1 alpha, apoptosis related protein Bax and caspase3 cutter expression, and B is a graph of quantitative analysis of A by Image J software.
Figure 8 shows a statistical plot of tumor volume change after treatment and tumor weight after treatment for each group of mice.
Figure 9 shows tumor cell apoptosis, angiogenesis and hypoxia analysis in tumor tissue after treatment of each group of mice. FIG. 10 shows changes in the angiogenesis and apoptosis-related proteins in tumor tissues after the end of treatment in mice of each group, wherein A shows changes in the expression of endothelial cell angiogenesis-related index p-ERK1/2, hypoxia index HIF1α, apoptosis-related protein Bax and C-caspase3 in tumor tissues detected by western blot, B shows a quantitative statistical graph of the protein expression amounts of p-ERK1/2 and ERK1/2 in A by Image J software, and C shows a statistical graph of the protein expression amounts of HIF1α, bax and C-caspase3 in A by Image J software.
FIG. 11 is a structural formula of an amphipathic molecule.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
The whole idea of the invention is as follows:
The current research proves that the growth and metastasis of the tumor are not only related to the proliferation of tumor cells, but also can not leave the support of peripheral blood vessels of the tumor. Tumor angiogenesis is the key to rapid growth, invasion and metastasis of tumors, and tumors continuously induce the generation of new blood vessels, which can also allow tumor cells to spread and metastasize along with blood flow in addition to providing nutrition and energy for tumor cell growth.
The applicant finds that the antitumor angiogenesis medicine can cut off the blood vessel supporting the tumor, so that the blood vessel is gradually reduced due to lack of oxygen and nutritional ingredients, the antitumor hypoxia prodrug is nontoxic, active medicine is generated through catalysis of reductase under the condition of hypoxia, the activity of topoisomerase II in tumor cells is inhibited, the tumor cells are further killed, the hypoxia condition in the tumor microenvironment is further aggravated by the antitumor medicine, and the capability of killing the tumor cells by the hypoxia prodrug is remarkably improved. The enhanced anti-tumor effect is realized by inhibiting tumor angiogenesis and simultaneously combining the hypoxia prodrug to kill tumor cells.
Because tumor tissues have special glycolytic metabolic processes, weak acidity of microenvironment is formed, based on the weak acidity, the invention provides an amphiphilic molecule for targeting and regulating tumor angiogenesis and an acid response type polypeptide nano-drug for targeting tumor tissues, synthesized anti-angiogenesis polypeptide molecules are used for grafting thioisocyanate functional molecules, self-assembly is carried out to form nano-particles, and the nano-particles are used as drug carriers to embed hypoxic anti-tumor drugs, such as AQ4N, TPZ, TH-302, car icotamide and the like (used as hypoxic prodrugs, and can be converted into anti-tumor active drugs under the condition of hypoxia), so as to improve the selective transportation of the drug AQ4N and the synergistic anti-tumor effect by using the bio-safe polypeptide as a carrier, and develop a new anti-tumor drug formulation for the current biological medicine field.
The amphiphilic molecule for targeting and regulating tumor angiogenesis and the acid response type polypeptide nano-drug for targeting tumor tissues are described in detail below by combining examples and experimental data.
Example 1
In this example, polypeptide nanomedicines were prepared by dissolving a small amount of amphiphilic molecules (structural formula shown in FIG. 11) in 10. Mu.L of dimethyl sulfoxide (DMSO), adding AQ4N to the above solution, mixing well, slowly dispersing the dimethyl sulfoxide solution with the dissolved polypeptide and AQ4N in 1mL of ultra pure water under 120W ultrasound, sonicating for 20min, standing for 1 hour at room temperature, and performing morphology and particle size characterization on the obtained nanomedicines (named ADF-NPs) using a transmission electron microscope. The polypeptide nano medicine without anti-vascular function (named ADW-NPs) is designed by using the same method by taking the nonfunctional amino acid sequence DW polypeptide as a control peptide. As shown in FIG. 1, the two nano-drugs prepared are spherical, have uniform particle sizes, and have average particle diameters of about 26.52nm and 23.19nm for ADF-NPs and ADW-NPspi, respectively.
Example 2:
The purpose of this example is to determine the acid response of a nano-drug.
The acid responsiveness is reflected by the change in morphology after the acid treatment. The polypeptide nano-drug obtained in example 1 was dispersed in PBS buffer at pH7.4 and pH 6.8. And observing the morphology change of the nano-drug under different pH environments by a transmission electron microscope. As shown in FIG. 2, the ADF-NPs and ADW-NPs of the nano-drugs all show the phenomenon that the nano-particles disintegrate and lose spherical structures in the environment of pH6.8, and the structural morphology is not changed obviously in the environment of pH 7.4. The nano-drug can disintegrate the spherical structure of the nano-drug through acid response, and is expected to realize the purpose of tumor microenvironment specific response release.
Example 3
The purpose of this example was to demonstrate the in vitro regulatory effect of the nano-drug prepared in example 1 on endothelial cells and the underlying mechanism of this effect.
HUVEC cells were first seeded into 6-well plates at a density of 2.5X10 4 cells/well, after the cells formed a fused monolayer, the tips of the sterile gun tips were perpendicular to the two parallel scratch wounds, washed 3 times with PBS, sequentially treated with DF, D-DF and ADF-NPs (pH all adjusted to acidic), photographed under a bright field microscope, recorded as 0h and marked at the photographing position, and placed in a cell incubator for continued culture for 24h. After 24 hours, photographs were taken at the same positions, and compared with scratch widths of 0 hours. The scratch width reflects the effect of different treatments on the ability to inhibit migration of HUVEC cells. Scratch width variation before and after different treatments was calculated using ImageJ software. The results are shown in FIG. 3, and the scratch width is obviously larger than that of the PBS group (Control group in FIG. 3) after the anti-angiogenesis polypeptide DF, the anti-angiogenesis polypeptide D-DF (amphiphilic molecule) modified by the acid response sequence and the ADF-NPs are treated, and the scratch width is obviously different from that of the PBS group in statistics. This suggests that D-DF and ADF-NPs are capable of significantly inhibiting migration of HUVEC cells. The mechanism by which this phenomenon occurs is then discussed. Expression of ERK and p-ERK proteins after various treatments was examined by WB (western blotting). The results are shown in FIG. 4, where ERK and p-ERK protein expression levels were significantly reduced after DF, D-DF, ADF-NPs treatment (GAPDH in the figure indicates an internal control to mark consistent protein loading for discrimination between high or low expression of other proteins) and there was a significant statistical difference compared to the PBS group. This suggests that the anti-angiogenic polypeptide inhibits endothelial cell migration by down regulating the expression of ERK and p-ERK proteins in the ERK pathway, and that neither modification of the anti-angiogenic polypeptide nor addition of AQ4N affects its own function.
Example 4
The purpose of this example was to verify the in vitro antitumor effect of the nano-drug prepared in example 1.
Tumor cells 4T1 were first seeded into 96-well plates at a density of 5 x 10 3 per well and allowed to attach overnight. Cells were then treated with AQ4N and ADF-NPs for 24 hours under itching and oxygen-enriched conditions. Cell viability was assessed using the CCK-8 kit. As shown in FIG. 5, AQ4N and ADF-NPs have more tumor cell killing activity under the anoxic condition. And ADF-NPs have better effect of killing tumor cells compared with free AQ 4N.
Next, the apoptosis of 4T1 cells by ADF-NPs was examined by apoptosis detection kit. First, 4T1 cells were inoculated into 6-well plates at a density of 2.5X10 4 cells/well, and placed in an incubator overnight, after growing to 50-60%, old medium in the well was removed, and after treatment with AQ4N and ADF-NPs under anoxic and oxygen-rich conditions, respectively, the cells were placed in the incubator for cultivation for 24 hours. After the culture is finished, the cells are collected and stained with an apoptosis kit, and are placed on a flow cytometer for detection. The results are shown in FIG. 6, in which the apoptosis rate of the AQ4N and ADF-NPs groups was about 37.5% under hypoxic conditions. This indicates that AQ4N is capable of significantly inducing apoptosis in tumor cells, and that the entrapment of the nano-drug does not affect its own effects.
Further, the possible action mechanism of tumor cell apoptosis after ADF-NPs treatment is verified by Western Blot experiments. By detecting the expression of related apoptosis proteins, the results are shown in FIG. 7, and compared with the PBS group, the ADF-NPs treated apoptosis-promoting proteins Bax and Cleaved-caspase-3 are obviously down-regulated (HIF 1 alpha represents hypoxia inducible factor 1 alpha), which is consistent with the results of cell killing experiments, and shows that ADF-NPs realize killing of tumor cells by regulating the expression of the related proteins. Taken together, these test results indicate that the nano-drug of example 1 has good in vitro anti-tumor effect.
Example 5
The purpose of this example was to verify the in vivo antitumor effect of the nano-drug prepared in example 1.
A certain amount of tumor cells 4T1 are injected subcutaneously into the right back of a BALB/c female mouse, and a breast cancer transplantation tumor model is established. The mice were then randomized into 6 groups of PBS, DF, D-DF, AQ4N, ADW-NPs and ADF-NPs, administered every other day seven times. Tumor volume changes of mice were monitored every 2 days throughout the treatment experiment, and the tumor volume calculation formula was tumor volume (mm 3) =0.5×width 2(mm2) ×length (mm), as shown in fig. 8A, in PBS group mice tumor volume was grown to 1000mm 3, and the other treatment groups uniformly inhibited tumor growth to some extent, while ADF-NPs treatment groups had more remarkable tumor growth inhibition effect than other groups. At the end of the treatment period, mice were euthanized, dissected and tumors were removed and weighed, as shown in the graph of FIG. 8B, with minimal tumor weight (average tumor weight: 0.37 g) in the mice treated with ADF-NPs treatment group and rapid tumor growth (average tumor weight: 1.38 g) in the PBS group, consistent with in vitro measurements.
Example 6
The aim of this example is to verify the potential mechanism of antitumor drug prepared in example 1.
As in example 5, after the treatment cycle, tumor tissue was removed and immunohistochemical immunofluorescent staining was performed on sections of tumor tissue to analyze the changes in TUNEL, CD31 and HIF-1. Alpha. In tumor tissue after various treatments.
The quantitative analysis of the green fluorescence intensity of TUNEL (apoptosis kit) staining for detecting apoptosis in tissues shows that after each group of treatment, certain phenomena for promoting tumor apoptosis are shown, wherein after ADF-NPs treatment, most of tumor cells are damaged at the tumor part, and statistical results show that large-area apoptosis occurs, and the results further verify that ADF-NPs has a strong tumor killing effect.
Through quantitative analysis of the CD31 positive area, the result shows that after treatment by the anti-angiogenesis polypeptide DF, the D-DF and the ADF-NPs as shown in FIG. 9B, the angiogenesis of tumor sites is obviously inhibited, and compared with a PBS group, the tumor sites have obvious statistical difference. While the rest of the groups have no obvious phenomenon.
The results of quantitative analysis of HIF-1α positive areas are shown in fig. 9C, and after anti-vascular treatment, positive areas are larger than those of PBS treatment groups, which shows that the tumor hypoxia state is aggravated by anti-vascular treatment, and that after combination with AQ4N, the tumor hypoxia state caused by anti-vascular treatment is relieved, which shows that by anti-vascular treatment, the combination of AQ4N reduces tumor blood supply by inhibiting angiogenesis on the one hand, inhibits growth of tumor cells, aggravates hypoxia on the other hand, so that the hypoxia prodrug AQ4N is more easily activated to AQ4 (an active form of AQ 4N) to kill tumor cells, wherein AQ4N itself is not toxic to cells, and the active drug AQ4 is generated by catalysis of reductase under the hypoxia condition, and the tumor hypoxia cells are killed by inhibiting topoisomerase II activity in the hypoxia cells.
The expression of related proteins in tumor tissues by each treatment group was then investigated by Western Blot experiments. The results are shown in FIG. 10. Through the anti-vascular treatment group, the expression level of ERK and p-ERK proteins in tumor tissues is obviously reduced, but the expression level of HIF-1 alpha is obviously enhanced, and after the combination of the treatment of AQ4N (ADF-NPs), the expression level of HIF-1 alpha is obviously reduced. After treatment of each group, the expression of the tumor-associated apoptosis proteins was increased to some extent. Among them, the expression of Bax and Cleaved-caspase-3 proteins in ADF-NPs treated groups had significant statistical differences compared to PBS group. These results also indicate that angiogenesis at the tumor site is inhibited by ERK pathway, but the hypoxia state at the tumor site is aggravated, and that better response of hypoxia prodrug is achieved by combining AQ4N, thereby achieving better killing effect of inducing apoptosis of tumor cells.
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (9)

1. An amphiphilic molecule for targeted regulation of tumor angiogenesis, which is characterized by comprising a hydrophilic end and a hydrophobic end, wherein the hydrophilic end comprises an anti-angiogenesis polypeptide and a connecting peptide, the N end of the anti-angiogenesis polypeptide is connected with the connecting peptide through a peptide bond, the N end of the connecting peptide is connected with 6-aminocaproic acid through an amide bond, the other end of the 6-aminocaproic acid is connected with the hydrophobic end through a thiourea bond, and the hydrophobic end is 3- (diethylamino) propyl isothiocyanate;
The amino acid sequence of the anti-angiogenesis polypeptide is PCAIWF, the amino acid sequence of the connecting peptide is DDEE, and the amino acid sequence of the hydrophilic end is DDEEPCAIWF.
2. Use of an amphiphilic molecule according to claim 1 for the preparation of a drug carrier or for the preparation of a drug for targeted modulation of tumor angiogenesis.
3. An acid response type polypeptide nano-drug for targeting tumor tissues, which is characterized by comprising a hydrophobic anti-tumor drug and a drug carrier coated outside the hydrophobic anti-tumor drug, wherein the drug carrier is the amphiphilic molecule of claim 1.
4. An acid responsive polypeptide nano-drug for targeting a tumor tissue according to claim 3, wherein said hydrophobic anti-tumor drug comprises a hypoxia prodrug.
5. The acid responsive polypeptide nano-drug targeting tumor tissue according to claim 3 or 4, wherein the hydrophobic anti-tumor drug comprises any one of AQ4N, TPZ, TH-302 and Caricotamide.
6. The acid responsive polypeptide nano-drug targeting tumor tissue according to claim 3 or 4, wherein the particle size of the polypeptide nano-drug is 25-30nm, and the average particle size is 26.52nm.
7. A method of preparing an acid responsive polypeptide nano-drug for targeting a tumor tissue according to claim 3, wherein the method of preparing comprises:
Co-dissolving amphiphilic molecules and hydrophobic antitumor drugs in an organic phase;
dispersing an organic phase dissolved with amphiphilic molecules and hydrophobic antitumor drugs in a water phase under an ultrasonic condition to obtain the polypeptide nano-drug.
8. The method of claim 7, wherein the organic phase comprises dimethyl sulfoxide and the aqueous phase comprises deionized water or phosphate buffer at ph 7.4.
9. The method for preparing the acid-responsive polypeptide nano-drug targeting tumor tissues according to claim 7, wherein the method is characterized in that an organic phase dissolved with amphiphilic molecules and hydrophobic antitumor drugs is dispersed in an aqueous phase under the ultrasonic condition to obtain the polypeptide nano-drug, and specifically comprises the following steps:
Dispersing the organic phase dissolved with amphiphilic molecules and hydrophobic antitumor drugs in the water phase under the action of ultrasonic waves with power of 80-120W, and standing for 1-4h for 10-30min to obtain the polypeptide nano-drugs.
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