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

Abstract

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, and 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 the release of 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 nanodrug targeting tumor tissue.

Description

Technical Field

[0001] The present invention belongs to the field of functional polypeptide nanotechnology, and particularly relates to an amphiphilic molecule for targeting and regulating tumor angiogenesis and an acid-responsive polypeptide nanomedicine targeting tumor tissue. Background Art

[0002] According to the World Cancer Report 2020 released by the World Health Organization, cancer has become the main killer threatening human health, and its mortality and morbidity rates continue to rise. The uncontrolled proliferation, invasiveness to surrounding tissue cells, and metastasis to other parts of the body are important characteristics of malignant tumors. The main clinical treatment strategies for malignant tumors are surgical resection and radiotherapy and chemotherapy. At present, the main problems facing tumor treatment are still the difficulty of transporting drugs to the tumor site and the problem of easy recurrence after treatment. Among many chemotherapy drugs, polypeptide drugs have unique advantages. Their drugability is higher than that of general chemical drugs. They have high biological activity, strong specificity, relatively weak toxic reactions, and are not easy to accumulate in the body. However, compared with small molecule chemical drugs, there are also shortcomings: polypeptide molecules have poor stability and are easily degraded in the body, so they have a short half-life and require continuous administration to maintain their efficacy, which brings inconvenience to patients.

[0003] Peptide nanomaterials are widely used in the in vivo delivery of drugs due to their passive targeting properties. Peptide nanoparticles have good biocompatibility, high safety, strong designability, certain targeting, and can protect peptide drugs from contact with proteases, thereby extending their half-life. Peptide self-assembly refers to a type of molecular aggregates or supramolecular structures with clear structure, stable structure, and certain physical and chemical properties formed by the spontaneous combination of amino acid residues through non-covalent bond interactions under appropriate conditions. Non-covalent bond interaction forces are the key to molecular self-assembly. Common non-covalent bond interaction forces include hydrogen bonding, van der Waals forces, electrostatic effects, hydrophobic effects, π-π stacking effects, cationic adsorption, etc. Non-covalent bond interaction forces maintain the structural stability and integrity of the self-assembly system. Summary of the invention

[0004] In order to solve the problem of poor efficacy of anti-tumor drugs, the present invention provides an amphiphilic molecule for targeted regulation of tumor angiogenesis. The amphiphilic molecule can be used as a drug carrier to prolong the half-life of anti-tumor drugs, and has a tumor site-specific response. It releases drugs in response to the weakly acidic microenvironment of the tumor, so that the drugs are efficiently transported to the tumor site. The amphiphilic molecule can also target and inhibit tumor angiogenesis, and synergistically improve the efficacy of anti-tumor drugs.

[0005] The present invention also provides an acid-responsive polypeptide nanomedicine targeting tumor tissue.

[0006] The present invention is achieved through the following technical solutions:

[0007] A targeted amphiphilic molecule for regulating tumor angiogenesis, the amphiphilic molecule comprising a hydrophilic end and a hydrophobic end, the hydrophilic end comprising an anti-angiogenesis polypeptide and a connecting peptide, the N-terminus of the anti-angiogenesis polypeptide being connected to the connecting peptide via a peptide bond, the N-terminus of the connecting peptide being connected to 6-aminocaproic acid via an amide bond, the other end of the 6-aminocaproic acid (Acp) being connected to the hydrophobic end via a thiourea bond, and the hydrophobic end being 3-(diethylamino)propyl isothiocyanate.

[0008] Furthermore, the amino acid sequence of the hydrophilic end is shown in SEQ ID NO.1.

[0009] 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.

[0010] Based on the same inventive concept, the present application also provides a use of an amphiphilic molecule for targeted regulation of tumor angiogenesis in the preparation of a drug carrier or a drug for targeted regulation of tumor angiogenesis.

[0011] Based on the same inventive concept, the present application also provides an acid-responsive polypeptide nanomedicine targeting tumor tissue, wherein the polypeptide nanomedicine comprises a hydrophobic anti-tumor drug and a drug carrier coated on the outside of the hydrophobic anti-tumor drug, wherein the drug carrier is an amphiphilic molecule that targets and regulates tumor angiogenesis.

[0012] Optionally, the hydrophobic anti-tumor drug comprises a hypoxic prodrug.

[0013] In the present application, the drug coated by the drug carrier is not limited to hypoxic prodrugs, but may also be other hydrophobic small molecule drugs or other hydrophobic anti-tumor drugs.

[0014] Optionally, the hydrophobic anti-tumor drug includes any one of AQ4N (banoxantrone), TPZ (Ti rapazamine / tirapazamine), TH-302 (Evofosfamide) and Car icotamide.

[0015] Furthermore, the particle size of the polypeptide nanomedicine is 25-30 nm, and the average particle size is 26.52 nm.

[0016] Based on the same inventive concept, the present application also provides a method for preparing an acid-responsive polypeptide nanodrug targeting tumor tissue, the preparation method comprising:

[0017] The amphiphilic molecule and the hydrophobic anti-tumor drug are co-dissolved in the organic phase;

[0018] The organic phase containing the amphiphilic molecules and the hydrophobic anti-tumor drug is dispersed in the aqueous phase under ultrasonic conditions to obtain the polypeptide nano-drug.

[0019] Furthermore, the organic phase includes dimethyl sulfoxide, and the aqueous phase includes deionized water or a phosphate buffer with a pH of 7.4.

[0020] Furthermore, under the ultrasonic condition, the organic phase containing the amphiphilic molecules and the hydrophobic anti-tumor drug is dispersed in the aqueous phase to obtain the polypeptide nano-drug, which specifically includes:

[0021] The organic phase containing the amphiphilic molecules and the hydrophobic anti-tumor drug is dispersed in the aqueous phase under the action of ultrasound with a power of 80-120 W, the ultrasound time is 10-30 minutes, and the mixture is allowed to stand for 1-4 hours to obtain the polypeptide nano-drug.

[0022] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0023] 1. The present invention discloses an amphiphilic molecule for targeted regulation of tumor angiogenesis, wherein the hydrophilic end of the amphiphilic molecule is an anti-angiogenesis polypeptide and a connecting peptide, and the hydrophobic end is 3-diethylaminopropyl isothiocyanate, and the hydrophilic end and the hydrophobic end are connected by (Acp). The amphiphilic molecule can be used as a drug carrier to prolong the half-life of anti-tumor drugs; and has a tumor site-specific response. In the slightly acidic environment of the tumor, due to the protonation of 3-(diethylamino)propyl isothiocyanate, the amphiphilic molecule responds to the acidic environment and depolymerizes, and the anti-tumor drug molecules are specifically released, so that the drug molecules are efficiently transported to the tumor site.

[0024] 2. The present invention discloses an amphiphilic molecule for targeted regulation of tumor angiogenesis. The amphiphilic molecule dissociates in response to an acidic environment to release anti-tumor drug molecules. Anti-angiogenesis polypeptides are also specifically released at the same time. The anti-angiogenesis polypeptides have the ability to target vascular endothelial growth factor receptors (VEGFRs) in tumor tissues, and after targeting, they antagonize VEGFRs, thereby regulating the ability of tumor-related angiogenesis. They can also target and inhibit tumor angiogenesis, thereby achieving a combined anti-tumor effect with drug molecules.

[0025] 3. The present invention discloses an amphiphilic molecule for targeted regulation of tumor angiogenesis, wherein the hydrophilic end and the hydrophobic end of the amphiphilic molecule are connected by (Acp), and 6-aminohexanoic acid is also called an alkyl spacer. In the synthesis reaction of the amphiphilic molecule, 3-(diethylamino)propyl isothiocyanate can react with -SH and the side chain -NH2 in the polypeptide structure, and the addition of Acp reduces the possibility of such side reactions. Acp provides a straight chain space of six carbons, which significantly reduces the steric hindrance of the reaction, improves the reaction efficiency, and reduces the difficulty of the reaction. In addition, when the anti-angiogenic polypeptide responds to the acidic environment of the tumor, it is usually accompanied by the removal of the last amino acid, and the introduction of the alkyl spacer Acp avoids this situation, thereby preventing the released polypeptide from being unable to effectively inhibit the angiogenesis of tumors. The connecting peptide plays a connecting role on the one hand, and can increase the hydrophilicity of the hydrophilic end on the other hand.

[0026] 4. The present invention discloses an acid-responsive polypeptide nanodrug targeting tumor tissues, wherein the polypeptide nanodrug comprises a hydrophobic antitumor drug and a drug carrier coated on the outside, i.e., an amphiphilic molecule. Compared with a single antitumor drug molecule, the polypeptide nanodrug has a longer half-life and can release antitumor drug molecules and anti-angiogenesis polypeptides in a targeted manner in the slightly acidic environment of the tumor, thereby achieving efficient transportation of antitumor drug molecules to the tumor site. At the same time, the anti-angiogenesis polypeptide can also inhibit angiogenesis in the tumor site, causing the tumor tissue to gradually shrink due to lack of oxygen and nutrients, thereby achieving a synergistically enhanced antitumor effect with the hydrophobic antitumor drug.

[0027] 5. The present invention discloses an acid-responsive polypeptide nanodrug targeting tumor tissue, wherein the hydrophobic anti-tumor drug can be a hypoxic prodrug, such as AQ4N. This type of hypoxic prodrug is non-toxic in itself and generates active drugs through reductase catalysis under hypoxic conditions, inhibiting the activity of topoisomerase II in tumor cells, thereby killing tumor cells. The anti-angiogenic polypeptide released in response to the tumor of the present invention further aggravates the hypoxia in the tumor microenvironment by inhibiting angiogenesis, and significantly improves the ability of hypoxic prodrugs to kill tumor cells. The polypeptide nanodrug of the present invention achieves a synergistically enhanced anti-tumor effect by inhibiting tumor angiogenesis and combining with hypoxic prodrugs to kill tumor cells.

[0028] 6. The present invention discloses an acid-responsive polypeptide nanodrug targeting tumor tissues. The polypeptide nanodrug is co-assembled with amphiphilic molecules and hydrophobic anti-tumor drugs under ultrasonic conditions to form a highly ordered nanostructure. The average particle size of the polypeptide nanodrug is 26.52 nm, which is small and helps tumor cell endocytosis. It has in vivo stability and is not easily degraded. No covalent bonds are generated during the self-assembly process, and there is no reverse reaction. The product has the characteristics of good biocompatibility and high safety, and is expected to become a good alternative biomedical material. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0030] Figure 1 This is a biological transmission electron microscope image of the prepared polypeptide nanomedicine under physiological conditions.

[0031] Figure 2 Biological transmission electron microscopy images of the prepared polypeptide nanomedicine under pH 7.4 and pH 6.8 conditions.

[0032] Figure 3 Images (Part A) and statistical analysis (Part B) of the effect of the prepared polypeptide nanomedicine on the migration ability of HUVEC cells.

[0033] Figure 4 To explore the potential mechanism of the prepared polypeptide nanomedicine in inhibiting HUVEC cell migration: wherein, A represents the expression of protein ERK1 / 2 and p-ERK1 / 2 detected by protein immunoblotting, and B represents the quantitative results of Figure A using Image J software.

[0034] Figure 5 The effect of the prepared polypeptide nanomedicine on the vitality of tumor cells under hypoxic and oxygen-rich conditions.

[0035] Figure 6 The effect of the prepared polypeptide nanomedicine on promoting tumor cell apoptosis under hypoxic and oxygen-rich conditions: wherein, A represents the effect of different treatments on cell apoptosis detected by flow cytometry, and B represents the statistical result of the cell apoptosis ratio in Figure A.

[0036] Figure 7 The potential mechanism of the prepared polypeptide nanomedicine in promoting tumor cell apoptosis under hypoxic and oxygen-rich conditions is analyzed: Figure A shows that Western Blot was used to detect the expression of hypoxia indicator HIF1α, apoptosis-related protein Bax and caspase3 splicing body, and Figure B is a quantitative analysis of Figure A using Image J software.

[0037] Figure 8 It shows the changes in tumor volume and the statistical graph of tumor weight of each group of mice after the end of treatment.

[0038] Fig. 9 It represents the analysis of tumor cell apoptosis, angiogenesis and hypoxia in the tumor tissue of each group of mice after the treatment. Fig.10 Figure 3 represents the expression changes of angiogenesis and apoptosis-related proteins in tumor tissues of mice in each group after treatment: A represents the expression changes 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 represents the quantitative statistical graph of the protein expression levels of p-ERK1 / 2 and ERK1 / 2 in Figure A using Image J software; C represents the statistical graph of the protein expression levels of HIF1α, Bax and C-caspase3 in Figure A using Image J software.

[0039] Fig.11 It is the structural formula of an amphiphilic molecule. DETAILED DESCRIPTION

[0040] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0041] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0042] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0043] The overall idea of ​​the present invention is as follows:

[0044] Current research has confirmed that tumor growth and metastasis are not only related to the proliferation of tumor cells themselves, but also inseparable from the support of blood vessels around the tumor. Tumor angiogenesis is the key to the rapid growth, invasion and metastasis of tumors. Tumors continuously induce the generation of new blood vessels, which not only provide nutrients and energy for tumor cell growth, but also allow tumor cells to spread and metastasize along the blood flow.

[0045] The applicant found that anti-tumor angiogenesis drugs can cut off the blood vessels supporting tumors, causing them to gradually shrink due to lack of oxygen and nutrients, which is the key to anti-tumor. Anti-tumor hypoxic prodrugs are non-toxic in themselves. Under hypoxic conditions, they are catalyzed by reductase to generate active drugs, inhibiting the activity of topoisomerase II in tumor cells, thereby killing tumor cells. Anti-angiogenesis drugs further aggravate the hypoxia in the tumor microenvironment and significantly improve the ability of hypoxic prodrugs to kill tumor cells. By inhibiting tumor angiogenesis and killing tumor cells in combination with hypoxic prodrugs, enhanced anti-tumor effects are achieved.

[0046] Since tumor tissue has a special glycolysis metabolic process, thus forming a weakly acidic microenvironment, based on this, the present invention provides an amphiphilic molecule for targeted regulation of tumor angiogenesis and an acid-responsive polypeptide nanomedicine targeting tumor tissue, using synthetic anti-angiogenesis polypeptide molecules to graft thioisocyanate functional molecules, self-assemble to form nanoparticles, and use them as drug carriers to embed hypoxic anti-tumor drugs, such as AQ4N, TPZ, TH-302 and Car icotamide (as hypoxic prodrugs, they are non-toxic themselves and can be converted into anti-tumor active drugs under hypoxic conditions), in order to use biosafe polypeptides as carriers to improve the selective transport of the drug AQ4N and the synergistic anti-tumor efficacy, and develop a new dosage form of anti-tumor drugs for the current biomedicine field.

[0047] The following will describe in detail an amphiphilic molecule for targeted regulation of tumor angiogenesis and an acid-responsive polypeptide nanomedicine targeting tumor tissues in combination with the embodiments and experimental data.

[0048] Example 1

[0049] In this example, the polypeptide nanomedicine is prepared by the following method: a small amount of amphiphilic molecules (structural formula: Fig.11 ) was dissolved in 10 μL of dimethyl sulfoxide (DMSO), and then AQ4N was added to the above solution and mixed. The dimethyl sulfoxide solution containing the polypeptide and AQ4N was slowly dispersed in 1 mL of ultrapure water under 120W ultrasonic conditions, ultrasonicated for 20 minutes, and allowed to stand at room temperature for 1 hour. The morphology and particle size of the obtained nanomedicine (named ADF-NPs) were characterized by transmission electron microscopy. The non-functional amino acid sequence DW polypeptide was used as a control peptide, and the same method was used to design a polypeptide nanomedicine without anti-vascular function (named ADW-NPs) as a control. The results are as follows Figure 1 As shown, the two prepared nanomedicines are spherical and have relatively uniform particle sizes. The average particle sizes of ADF-NPs and ADW-NPspi are approximately 26.52nm and 23.19nm, respectively.

[0050] Embodiment 2:

[0051] The purpose of this example is to determine the acid response of nanomedicine.

[0052] The acid responsiveness is reflected by the morphological changes after acid treatment. The polypeptide nanodrug obtained in Example 1 was dispersed in PBS buffer at pH 7.4 and pH 6.8. The morphological changes of the nanodrug under different pH environments were observed by transmission electron microscopy. The results are shown in FIG. Figure 2 As shown in the figure, nanomedicines ADF-NPs and ADW-NPs showed nanoparticle disintegration and loss of spherical structure under pH 6.8, while there was no obvious change in the structure morphology under pH 7.4. This shows that the nanomedicines involved can disintegrate the spherical structure of the nanomedicine through acid response, which is expected to achieve the purpose of tumor microenvironment-specific response release.

[0053] Example 3

[0054] The purpose of this example is to verify the in vitro regulatory effect of the nanomedicine prepared in Example 1 on endothelial cells and explore the potential mechanism of this effect.

[0055] First, HUVEC cells were cultured at 2.5×10 4 The cells were inoculated at a density of 100 cells / well in a 6-well plate. After the cells formed a confluent monolayer, a 200 μL sterile pipette tip was used to perpendicularly scratch two parallel wounds. The cells were washed three times with PBS and treated with DF, D-DF and ADF-NPs (pH was adjusted to acidic) in sequence. Pictures were taken under a bright field microscope, recorded as 0 h and the position of the picture was marked. The cells were placed in a cell culture incubator and cultured for 24 h. After 24 h, pictures were taken at the same position and compared with the scratch width at 0 h. The scratch width reflects the effect of different treatments on the inhibition of the migration ability of HUVEC cells. ImageJ software was used to calculate the changes in scratch width before and after different treatments. The results are shown in the figure. Figure 3 As shown in Figure 2, the scratch width after treatment with anti-angiogenic peptide DF, anti-angiogenic peptide D-DF (i.e., amphiphilic molecule) modified with acid-responsive sequence, and ADF-NPs was significantly greater than that of the PBS group ( Figure 3 The difference between D-DF and ADF-NPs and HUVEC cells was statistically significant (P < 0.05). This indicates that D-DF and ADF-NPs can significantly inhibit the migration of HUVEC cells. The mechanism of this phenomenon was then explored. The expression of ERK and p-ERK proteins after different treatments was detected by WB (protein immunoblotting). The results are shown in Figure 2. Figure 4As shown in the figure, after treatment with DF, D-DF, and ADF-NPs, the expression of ERK and p-ERK proteins was significantly reduced (GAPDH in the figure represents the internal reference, which is used to calibrate the protein loading amount to be consistent and to distinguish the high or low expression of other proteins), and there was a significant statistical difference compared with the PBS group. This indicates that the anti-angiogenic peptide inhibits the migration behavior of endothelial cells by downregulating the expression of ERK and p-ERK proteins in the ERK pathway, and that the modification of the anti-angiogenic peptide or the addition of AQ4N does not affect its own function.

[0056] Example 4

[0057] The purpose of this example is to verify the in vitro anti-tumor effect of the nanomedicine prepared in Example 1.

[0058] First, 4T1 tumor cells were cultured at 5 × 10 3 The cells were seeded at a density of 100 μg / ml in 96-well plates and allowed to attach overnight. The cells were then treated with AQ4N and ADF-NPs for 24 h under hypoxic and oxygen-rich conditions. Cell viability was assessed using the CCK-8 kit. Figure 5 As shown in the figure, under hypoxic conditions, AQ4N and ADF-NPs have more activity in killing tumor cells. And ADF-NPs has a better effect in killing tumor cells than free AQ4N.

[0059] Then, the apoptosis of 4T1 cells by ADF-NPs was investigated using a cell apoptosis detection kit. 4 The cells were seeded at a density of 100 cells / well in a 6-well plate and placed in an incubator overnight. When the cells grew to 50-60%, the old culture medium in the well was removed. The cells were treated with AQ4N and ADF-NPs under hypoxic and oxygen-rich conditions, respectively, and then placed in an incubator for 24 hours. After the culture was completed, the cells were collected and stained with a cell apoptosis kit and tested on a flow cytometer. The results are shown in Figure 2. Figure 6 As shown in the figure, under hypoxic conditions, the apoptosis rate of the AQ4N and ADF-NPs groups was about 37.5%, which indicates that AQ4N can significantly induce apoptosis of tumor cells, and the encapsulation of nanomedicine does not affect its own effects.

[0060] Furthermore, Western Blot experiments were performed to verify the possible mechanism of tumor cell apoptosis after ADF-NPs treatment. The expression of related apoptosis proteins was detected. Figure 7As shown, compared with the PBS group, the apoptosis-promoting proteins Bax and Cleaved-caspase-3 after ADF-NPs treatment were significantly downregulated (HIF1α represents hypoxia-inducible factor 1α), which is consistent with the results of the cell killing experiment, indicating that ADF-NPs kills tumor cells by regulating the expression of these related proteins. In summary, these experimental results show that the nanomedicine in Example 1 has a good in vitro anti-tumor effect.

[0061] Example 5

[0062] The purpose of this example is to verify the in vivo anti-tumor effect of the nanomedicine prepared in Example 1.

[0063] First, a certain amount of tumor cells 4T1 were subcutaneously injected into the right back of BALB / c female mice to establish a breast cancer transplant tumor model. Then the mice were randomly divided into 6 groups: PBS, DF, D-DF, AQ4N, ADW-NPs and ADF-NPs groups, and the drugs were given every other day for a total of seven times. During the entire treatment experiment, the tumor volume changes of the mice were monitored every 2 days. The tumor volume calculation formula is: tumor volume (mm 3 )=0.5×width 2 (mm 2 )×length (mm), such as Figure 8 As shown in Figure A, the tumor volume of mice in the PBS group grew to 1000mm 3 , while the other treatment groups all inhibited tumor growth to a certain extent, and the ADF-NPs treatment group had a more significant inhibitory effect on tumor growth than the other groups. After the treatment cycle, the mice were euthanized, the mice were dissected, and the tumors were removed and weighed. Figure 8 As shown in Figure B, consistent with the in vitro measurement results, the tumor weight of mice treated with ADF-NPs was the smallest (average tumor weight: 0.37 g), and the tumor in the PBS group grew rapidly (average tumor weight: 1.38 g).

[0064] Example 6

[0065] The purpose of this example is to verify the potential anti-tumor mechanism of the nanomedicine prepared in Example 1.

[0066] Similar to Example 5, after the treatment cycle, the tumor tissue was removed, and the tumor tissue sections were subjected to immunohistochemical and immunofluorescent staining to analyze the changes in TUNEL, CD31 and HIF-1α in the tumor tissue after different treatments.

[0067] The results are as follows: Fig. 9As shown in A, after treatment in each group, a certain phenomenon of promoting tumor apoptosis was shown. After ADF-NPs treatment, most of the tumor cells were damaged in the tumor site. Statistical results showed that a large area of ​​apoptosis occurred. These results further verified that ADF-NPs had a strong tumor killing effect.

[0068] The results of quantitative analysis of CD31 positive area were as follows: Fig. 9 As shown in B, after treatment with anti-angiogenic peptides DF, D-DF and ADF-NPs, angiogenesis in the tumor site was significantly inhibited, with significant statistical differences compared with the PBS group. There was no significant phenomenon in the other groups.

[0069] By quantitative analysis of the HIF-1α positive area, the results are as follows Fig. 9 As shown in C, after anti-vascular treatment, the positive area was larger than that in the PBS treatment group, indicating that the hypoxic state of the tumor was aggravated by anti-vascular treatment. After combined with AQ4N, the hypoxic state of the tumor caused by anti-vascular treatment was alleviated. This shows that the combination of anti-vascular treatment with AQ4N can reduce tumor blood supply and inhibit tumor cell growth by inhibiting angiogenesis, and aggravate hypoxia, making the hypoxic prodrug AQ4N more easily activated to AQ4 (the active form of AQ4N) to kill tumor cells. Among them, AQ4N itself is not toxic to cells. Under hypoxic conditions, it is catalyzed by reductase to generate active drug AQ4, which kills tumor hypoxic cells by inhibiting the activity of topoisomerase II in hypoxic cells.

[0070] Then, Western Blot experiments were performed to explore the expression of related proteins in tumor tissues in each treatment group. Fig.10 As shown. After anti-vascular treatment, the expression of ERK and p-ERK proteins in tumor tissues was significantly reduced, but the expression of HIF-1α was significantly enhanced. After combined treatment with AQ4N (ADF-NPs), the expression of HIF-1α was significantly weakened. After treatment in each group, the expression of tumor-related apoptosis proteins increased to a certain extent. Among them, the expression of Bax and Cleaved-caspase-3 proteins in the ADF-NPs treatment group was significantly different from that in the PBS group. These results also show that angiogenesis in the tumor site is inhibited through the ERK pathway, but the hypoxia state in the tumor site is aggravated, while a better response to hypoxia prodrugs is achieved by combining with AQ4N, achieving a better killing effect of inducing tumor cell apoptosis.

[0071] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0072] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0073] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. An amphiphilic molecule for targeted regulation of tumor angiogenesis, characterized in that: The amphiphilic molecule comprises a hydrophilic end and a hydrophobic end, the hydrophilic end comprises an anti-angiogenic polypeptide and a connecting peptide, the N-terminus of the anti-angiogenic polypeptide is connected to the connecting peptide via a peptide bond, the N-terminus of the connecting peptide is connected to 6-aminocaproic acid via an amide bond, the other end of the 6-aminocaproic acid is connected to the hydrophobic end via 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 the amphiphilic molecule according to claim 1 in preparing a drug carrier or a drug for targeted regulation of tumor angiogenesis.

3. An acid-responsive polypeptide nanodrug targeting tumor tissue, characterized in that: The polypeptide nanomedicine comprises a hydrophobic anti-tumor drug and a drug carrier coated on the outside of the hydrophobic anti-tumor drug, and the drug carrier is the amphiphilic molecule as claimed in claim 1.

4. The acid-responsive polypeptide nanodrug targeting tumor tissue according to claim 3, characterized in that: The hydrophobic anti-tumor drug includes a hypoxic prodrug.

5. The acid-responsive polypeptide nanodrug targeting tumor tissue according to claim 3 or 4, characterized in that: The hydrophobic anti-tumor drug includes any one of AQ4N, TPZ, TH-302 and Caricotamide.

6. The acid-responsive polypeptide nanodrug targeting tumor tissue according to claim 3 or 4, characterized in that: The particle size of the polypeptide nano drug is 25-30 nm, and the average particle size is 26.52 nm.

7. A method for preparing the acid-responsive polypeptide nanodrug targeting tumor tissue as claimed in claim 3, characterized in that: The preparation method comprises: The amphiphilic molecule and the hydrophobic anti-tumor drug are co-dissolved in the organic phase; The organic phase containing the amphiphilic molecules and the hydrophobic anti-tumor drug is dispersed in the aqueous phase under ultrasonic conditions to obtain the polypeptide nano-drug.

8. The method for preparing an acid-responsive polypeptide nanodrug targeting tumor tissue according to claim 7, characterized in that: The organic phase includes dimethyl sulfoxide, and the aqueous phase includes deionized water or a phosphate buffer with a pH of 7.

4.

9. The method for preparing an acid-responsive polypeptide nanodrug targeting tumor tissue according to claim 7, characterized in that: The method of dispersing the organic phase containing the amphiphilic molecules and the hydrophobic anti-tumor drug in the aqueous phase under the ultrasonic condition to obtain the polypeptide nano-drug specifically comprises: The organic phase containing the amphiphilic molecules and the hydrophobic anti-tumor drug is dispersed in the aqueous phase under the action of ultrasound with a power of 80-120 W, the ultrasound time is 10-30 minutes, and the mixture is allowed to stand for 1-4 hours to obtain the polypeptide nano-drug.

Images