Block copolypeptides and use thereof

Abstract

This invention provides a block copolymer comprising a first positively charged peptide fragment consisting of 5 to 20 substituted or unsubstituted L-lysine, L-arginine, L-ornithine, or L-homogeneous acid. This block copolymer can inhibit the migration and replication of cancer cells, reduce tumor growth, and regulate the gut microbiota, specifically increasing the population of *Akkermansia muciniphila*. This invention also provides a method for treating cancer and a method for increasing the proportion of *Akkermansia muciniphila* (Akk) in the gut, comprising administering an effective amount of the block copolymer to a subject in need.

Description

Technical Field

[0001] sequence list

[0002] Pursuant to 37 CFR § 1.831-835, this application contains a computer-readable sequence listing, which has been electronically submitted in XML format and is incorporated herein by reference in its entirety. The XML file was created on December 23, 2022, named 21142US sequence listing.xml, and has a size of 16.4kb.

[0003] This disclosure relates to block copolypeptides, particularly block copolypeptides comprising a first positively charged peptide fragment and a second hydrophobic peptide fragment, which can be used to inhibit cancer cell migration and replication, reduce tumor growth, and regulate the gut microbiota. These block copolypeptides can also serve as gene vectors to improve transfection efficiency, endosomal escape, and biocompatibility. Prior Technology

[0004] Peptide drugs, situated between small molecule and protein drugs, possess unique biochemical and therapeutic properties that distinguish them from the other two extremes and sometimes give them advantages over other drugs. Peptide drugs have lower toxicity, higher target specificity than most chemical drugs, and are easier to synthesize than protein drugs. Nevertheless, the development of peptide drugs has had a significant impact on clinical medicine over the past 20 years; more than 80 peptide drugs have been approved, and hundreds more are in preclinical and clinical trials worldwide. However, peptides suffer from short in vivo half-lives, sometimes only a few minutes, often rendering their natural form unsuitable for therapeutic administration. Therefore, the field of technology needs modified therapeutic peptides with longer half-lives and / or reduced clearance rates compared to unmodified therapeutic peptides, as well as additional therapeutic advantages.

[0005] Furthermore, gene therapy is a radical treatment for patients with gene-related defects, and its success is directly linked to the efficiency of the gene vector. Currently, two distinguishable forms of gene vectors are used: viral vectors and non-viral vectors. Viral vectors are considered to carry a higher risk due to the potential for adverse immune responses or sequelae. Non-viral vectors, such as cationic polymers (containing peptides), have fewer side effects but typically have poor transfection efficiency. It has been reported that increasing the amphiphilicity of cationic polymer modifications, including facile branching modifications, can improve transfection efficiency. Summary of the Invention

[0006] In view of the aforementioned deficiencies, this disclosure is based on the discovery of the potential of a block copolymer peptide comprising a first positively charged peptide fragment and a second hydrophobic peptide fragment, which inhibits cancer cell migration and attachment by disrupting the integrity of the cancer cell membrane and interfering with the folding of epithelial cadherins on the cancer cell membrane. Furthermore, this disclosure reveals that the block copolymer peptide comprising the first positively charged peptide fragment and the second hydrophobic peptide fragment, as a gene vector, exhibits improved transfection efficiency, endosome detachment, and biocompatibility.

[0007] The metastasis process of cancer cells mainly consists of four steps: i) separation from the primary tumor, ii) migration to the blood or lymphatic circulation, iii) attachment and adaptation to secondary sites, and iv) transformation / growth into new metastatic tumors. Epithelial cadherin plays an important role in the separation, systemic dissemination, and metastatic stages of invasive ductal carcinoma.

[0008] Many metastatic tumors exhibit high levels of epithelial cadherin, as epithelial cells exhibiting epithelial cadherin can become more invasive and metastasize without the involvement of other transforming molecules. Notably, the conformation of epithelial cadherin is only stable when Ca2+ binds to its negatively charged extracellular motif. Amino acid derivatives with cationic and large functional groups inhibit the formation of intermediates during the dimerization of P-cadherin. The cationic group may interact with the negatively charged region located near the cavity between the EC1 and EC2 domains of P-cadherin, while the large functional groups, such as indole or benzyl, can clamp into this interface to hinder dimerization. Therefore, designing peptides that inhibit cell attachment by interfering with the folding of epithelial cadherin using similar strategies is a starting point for the development of short peptides.

[0009] Essentially, cancer cell membranes exhibit a greater proportion of anionic components than normal cells, and this property is being used to develop cationic hydrophobic peptides as anticancer agents. Furthermore, the design of anticancer peptides (ACPs) has incorporated structures that influence cell membrane integrity or protein-protein interactions, such as α-helices, β-lamellae, and random coils.

[0010] Thus, in the first state, this disclosure provides a block copolymer peptide comprising a first positively charged peptide fragment and a second hydrophobic peptide fragment for cancer therapy.

[0011] There are no particular restrictions on the amino acids used to construct positively charged or hydrophobic segments, and they can be all natural amino acids, all non-natural amino acids, or a combination of both. Similarly, there are no particular restrictions on the sequence of amino acids in each segment, as long as the segment composed of amino acids exhibits the desired property, i.e., positive charge or hydrophobicity. Furthermore, there are no particular restrictions on the order of the segments.

[0012] In at least one embodiment disclosed herein, the first positively charged peptide fragment is composed of L-lysine, L-arginine, L-ornithine, or L-hoarginine, and the second hydrophobic peptide fragment is composed of L-phenylalanine, L-tryptophan, L-DOPA, L-tyrosine, L-cysteine, S-benzyl-L-cysteine, or S-methylcysteine, wherein the first peptide fragment and the second peptide fragment each comprise 5 to 20 amino acids.

[0013] The first peptide fragment and the second peptide fragment may each comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. The total length of the block copolymer disclosed herein can therefore be from 10 to 40 amino acids, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.

[0014] The first positively charged peptide fragment may consist of only L-lysine, only L-arginine, only L-ornithine, only L-holysine, or both or all of them. In at least one embodiment, the first positively charged peptide fragment is composed of L-lysine.

[0015] The second hydrophobic peptide fragment may be composed of only L-phenylalanine, only L-tryptophan, only L-DOPA, only L-tyrosine, only L-cysteine, only S-benzyl-L-cysteine, only S-methylcysteine, or both, three, or more thereof. In at least one embodiment, the second hydrophobic peptide fragment is composed of only S-benzyl-L-cysteine ​​or only L-cysteine.

[0016] In at least one embodiment, the first positively charged peptide fragment is L-lysine and the second hydrophobic peptide fragment is L-cysteine ​​or S-benzyl-L-cysteine. In another embodiment, the block copolymer peptide comprises Lys5BnCys5 (SEQ ID NO:2), Lys5Cys5 (SEQ ID NO:1), Lys10BnCys5 (SEQ ID NO:5), Lys10Cys5 (SEQ ID NO:19), Lys15BnCys5 (SEQ ID NO:3), or Lys15Cys5 (SEQ ID NO:20).

[0017] In at least one embodiment, the block copolymer is a single chain, or branched, or star-shaped; for example, the block copolymer has the main structure shown in Figure 17. In Figure 17, each branch or arm of the branched or star-shaped block copolymer independently has the first positively charged peptide fragment and the second hydrophobic peptide fragment, but the invention is not limited to this approach. In one embodiment, one branch or arm has the first positively charged peptide fragment and another branch or arm has the second hydrophobic peptide fragment. In another embodiment, one branch or arm has both the first positively charged peptide fragment and the second hydrophobic peptide fragment; for example, the branched or star-shaped block copolymer is selected from one shown in Figure 20.

[0018] In at least one embodiment, the side chain of the block copolymer is modified with at least one of the group consisting of p-methoxybenzaldehyde, vanillin, cinnamaldehyde, catechol, indole, phenol, and phenyl, for example, the modified block copolymer has the main structure shown in Schematic 18.

[0019] In one embodiment, the block copolymer is star-shaped or has multiple arms, each arm comprising the first positively charged peptide fragment and the second hydrophobic peptide fragment. In one embodiment, the total number of arms is from 3 to 24, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0020] This application also provides a method for treating cancer, comprising administering an effective amount of a pharmaceutical composition to a subject in need, wherein the pharmaceutical composition comprises the block copolymer of claim 1 and a pharmaceutically acceptable carrier.

[0021] In at least one embodiment, the effective amount is from 5 mg / kg to 10 mg / kg body weight of the subject, for example, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 mg / kg body weight.

[0022] In at least one embodiment, the cancer is lung cancer, breast cancer, ovarian cancer, brain cancer, kidney cancer, oral cancer, gastric or esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, uterine cancer, endometrial cancer, cervical cancer, stomach cancer, skin cancer, testicular cancer, prostate cancer, or thyroid cancer. In one embodiment, the cancer is lung cancer.

[0023] In one embodiment, the block copolymer inhibits the migration and attachment of cancer cells. In another embodiment, the block copolymer inhibits the expression of epithelial cadherin in cancer cells. In yet another embodiment, the block copolymer inhibits both the migration and attachment of cancer cells and the expression of epithelial cadherin in cancer cells.

[0024] In at least one embodiment, the block copolymer is applied to the subject intravenously, intra-arterially, intraperitoneally, intramuscularly, intradermally, intratumorally, orally, transdermally, transnasally, transorally, transrectally, transvaginally, by inhalation, or by local application.

[0025] In at least one embodiment, the method of treating cancer further includes administering an effective amount of an anticancer agent to the subject.

[0026] In at least one embodiment, the anticancer agent may be, but is not limited to, cisplatin, oxaliplatin, nedaplatin, lobaplatin, or combinations thereof.

[0027] In one embodiment, the anticancer agent is administered simultaneously with the block copolymer. In another embodiment, the anticancer agent is administered before or after the block copolymer. In yet another embodiment, the anticancer agent and the block copolymer are administered via the same route. In yet another embodiment, the anticancer agent and the block copolymer are administered via a different route.

[0028] This disclosure also provides a method for increasing Akkermansia muciniphila (Akk) in the gut, comprising administering an effective amount of a pharmaceutical composition to a subject in need, wherein the pharmaceutical composition comprises the block copolymer of claim 1 and a pharmaceutically acceptable carrier.

[0029] Furthermore, this disclosure provides a pharmaceutical composition comprising a block copolymer and a pharmaceutically acceptable carrier. This disclosure also provides the use of the block copolymer in the preparation of a medicament for treating cancer. This invention provides a block copolymer for treating cancer.

[0030] In the second state, this disclosure also provides a gene vector, including a block copolymer comprising a first positively charged peptide fragment and a second hydrophobic peptide fragment. Simple Explanation of the Diagram

[0031] The invention will be more readily understood from the following description taken in conjunction with the accompanying drawings.

[0032] Figure 1 is a schematic diagram of the possible signaling pathways for the synthesis of Lys5Cys5 (SEQ ID NO:1) and Lys5BnCys5 (SEQ ID NO:2) in lung cancer cells.

[0033] Figures 2A to 2C show the effects of Lys5Cys5 (SEQ ID NO:1) or Lys5BnCys5 (SEQ ID NO:2) on cytotoxic and hemolytic activities. Figure 2A is a bar graph (n=4) showing the survival rate (%) of A549 cells treated with peptide Lys5Cys5 (SEQ ID NO:1) or Lys5BnCys5 (SEQ ID NO:2) for 24 hours in the presence or absence of cisplatin (CP, 5 μM), as determined by CCK-8 assay. Figure 2B is a bar graph (n=4) showing the survival rate (%) of H1299 cells treated with peptide (Lys5Cys5 (SEQ ID NO:1) or Lys5BnCys5 (SEQ ID NO:2) for 24 hours in the presence or absence of cisplatin (CP, 5 μM), as determined by CCK-8 assay. Figure 2C is a bar graph (n=4) showing the survival rate (%) of BEAS-2B cells treated with peptides (Lys5Cys5 (SEQ ID NO:1) or Lys5BnCys5 (SEQ ID NO:2)) for 24 hours, with or without cisplatin (CP, 5 μM), as determined by CCK-8 assay. Figure 2D is a bar graph (left column) (n=4) showing the percentage of hemolysis when analyzing heme in the supernatant using a microdisc reader at 405 nm, and a table showing this percentage of hemolysis (right column). The results were analyzed using two-way ANOVA (Figures 2A to 2D).

[0034] Figures 3A to 3C show the effects of Lys5Cys5 (SEQ ID NO:1) or Lys5BnCys5 (SEQ ID NO:2) on migration and adhesion in H1299 cells. Figure 3A shows the results of a scratch healing assay in H1299 cells, treated with Lys5Cys5 (SEQ ID NO:1), Lys5BnCys5 (SEQ ID NO:2), cisplatin, or peptide-bound cisplatin (CP, 5 μM) to determine cell migration ability. The peptide solution was added immediately after wound formation. The wound was assessed 24 hours after peptide application (scale bar = 50 μm). Figure 3B is a bar graph showing the percentage of wound healing area quantified based on the total cell area as 100% (n=3). Figure 3C is a bar graph showing the quantified adherent cells at the bottom of the well (n=3). 1 × 10⁵ cells / well were seeded into 12-well trays and treated with Lys₅Cys₅ (SEQ ID NO:1) or Lys₅BnCys₅ (SEQ ID NO:2) for 24 hours. The ability to attach to the solid phase was determined by CCK-8 assay.

[0035] Figures 4A and 4B show the changes in apoptosis signals and adhesion ability in lung cancer cells induced by Lys5Cys5 (SEQ ID NO:1) and Lys5BnCys5 (SEQ ID NO:2). Figure 4A shows the results of caspase-3-dependent apoptosis induced by Lys5Cys5 (SEQ ID NO:1) or Lys5BnCys5 (SEQ ID NO:2) in H1299 cells. Cells were treated with peptides for 24 hours, and the expression of epithelial cadherin, apoptosis-dependent caspase-3, and PARP in all cell lysate products was analyzed using immunoblot analysis. Histograms representing relative performance levels were used to quantify the relative performance levels using ImageJ software based on β-actin reference band normalization (n=3; *, #p<0.05, **, ##p<0.01, and ***, ###p<0.001). The asterisks and # symbols indicate that the group treated with apoptosis protein 3 compared to PARP-3 was statistically significant. Figure 4B shows the fluorescence images (scale bar = 100 μm) of one group of A549 and H1299 cells treated with Lys5Cys5 (SEQ ID NO:1) or Lys5BnCys5 (SEQ ID NO:2) (4 μM) for 24 hours using an inverted fluorescence microscope. White arrows indicate chromatin condensation in the cell nucleus.

[0036] Figures 5A to 5F show the results of Lys5BnCys5 (SEQ ID NO:2) inhibiting LL2 tumor growth and prolonging survival in tumor-bearing mice. Figure 5A shows a group of bioluminescent images of mice injected with D-luciferin on day 12, and displays a whole-body map and photon flux scale bar. Figure 5B shows a bar graph of quantitative bioluminescent imaging using the IVIS-200 system. Figure 5C is a set of graphs of LLC tumor-bearing mice treated with saline, Lys5BnCys5 (SEQ ID NO:2) (5 mg / kg), Lys5BnCys5 (10 mg / kg), Lys5BnCys5 (5 mg / kg) + cisplatin (2 mg / kg), and cisplatin (2 mg / kg) on ​​days 7, 10, 16, and 22 after the first treatment (n=8 to 10; *p<0.05; **p<0.01 and ***p<0.001). Figure 5D shows the changes in tumor mass in LLC tumor-bearing NOD-SCID mice after intratumoral injection of saline, Lys5BnCys5 (SEQ ID NO:2) (5 mg / kg), Lys5BnCys5 (10 mg / kg), Lys5BnCys5 (5 mg / kg) + cisplatin (2 mg / kg), and cisplatin (2 mg / kg) (n=8). Figure 5E shows the changes in body weight recorded from day 0 (n=8). Figure 5F shows the Kaplan-Meier survival curves of the median survival time of tumor-bearing mice under different treatments. The log-rank test was used to assess differences in survival rates (n=8) (b: unpaired Student t-test; d, e: two-factor variance analysis).

[0037] Figures 6A through 6D show the results of Lys5BnCys5 (SEQ ID NO:2) reducing LL2 tumor metastasis in the lungs. Figure 6A is a graph of mouse tumors. Figure 6B shows the results of measuring the size and weight of tumors removed on day 32. Figures 6C and D show the results of analyzing the number of metastatic lung nodules using the lung tumor colony formation assay indicated by black arrows. Scale bar = 200 μm (n = 8) (b and c: unpaired Student t-test).

[0038] Figures 7A to 7C show the microbial diversity of the different treatment groups. Figure 7A shows that the Lys5BnCys5 (SEQ ID NO:2) treatment exhibited significant α-diversity (Shannon index) compared to the saline treatment and the Lys5BnCys5+cisplatin treatment. Figure 7B shows the β-diversity from principal coordinate analysis (PCoA), demonstrating significant differences among these treatment groups in the total microbial composition. Figure 7C shows the relative abundance of rational taxonomic units (OTUs) at the phylum and species levels in these groups (saline, Lys5BnCys5 (SEQ ID NO:2), and Lys5BnCys5+cisplatin treatment groups).

[0039] Figures 8A to 8D show heatmap analyses of specific microbial components in the Lys5BnCys5 (SEQ ID NO:2) treatment group. Figures 8A and 8B show that the Lys5BnCys5 (SEQ ID NO:2) treatment increased the level of Verrucomicrobia compared to the pre-treatment and saline groups. Figures 8C and 8D show that the Lys5BnCys5 (SEQ ID NO:2) treatment increased the relative proportion of Ekmanophilus compared to the pre-treatment and saline groups.

[0040] Figure 9 is a schematic diagram of the interaction of Lys5BnCys5 (SEQ ID NO:2) peptides on cancer cell membranes and the analysis of gut microbiota after treatment with Lys5BnCys5 (SEQ ID NO:2) peptides.

[0041] Figures 10A to 10C show the 1H NMR spectra of (a) Z-Lys5, (b) Z-Lys5BnCys5, and (c) Lys5BnCys5 (SEQ ID NO:2) peptides dissolved in TFA-d1, TFA-d1, and DMSO-d6, respectively.

[0042] Figures 11A and 11B show the 1H NMR spectra of (a) Lys5BnCys5 (SEQ ID NO:2) and (b) Lys5Cys5 (SEQ ID NO:1) peptides dissolved in TFA-d1 and DMSO-d6, respectively.

[0043] Figure 12A shows the circular dichroism (CD) spectra of the Lys5BnCys5 (SEQ ID NO:2) and Lys5Cys5 (SEQ ID NO:1) peptides. For CD analysis, the peptide concentration was 0.1 mg mL⁻¹ in DI water. Figure 12B shows the FTIR spectra of the Lys5BnCys5 (SEQ ID NO:2) and Lys5Cys5 (SEQ ID NO:1) peptides.

[0044] Figure 13 is a dot plot showing the small-angle X-ray scattering (SAXS) pattern of the Lys5BnCys5 (SEQ ID NO:2) peptide at a concentration of 0.5 wt% in DI water.

[0045] Figures 14A and 14B are dot plots showing the pyrene emission I3 / I1 values ​​of (a) Lys5BnCys5 (SEQ ID NO:2) and (b) Lys5Cys5 (SEQ ID NO:1) peptides as a function of peptide concentration. The I3 / I1 value is reduced by 0.65.

[0046] Figure 15 shows a series of inverted fluorescence micrographs (scale bar = 100 μm) of A549 and H1299 cells treated with peptide (4 μM) for 6 hours and stained with 1,1'-diethyl-4,4'-carbocyanine iodide (DCI), 1 μg / mL.

[0047] Figure 16 shows a series of immunochemical staining images of H1299 cells treated with Lys5Cys5 (SEQ ID NO:1) or Lys5BnCys5 (SEQ ID NO:2) for 24 hours and stained with anti-epithelial cadherin antibody (brown) and counterstained with hematoxylin (blue). CP: cisplatin (5 μM). Scale bar = 100 μm.

[0048] Figure 17 shows the structures of linear and star-shaped block and graft copolymers.

[0049] Figure 18 shows the structures of linear and star-shaped block and graft copolymers.

[0050] Figure 19 shows the cell survival rate (%) at various concentrations of different copolypeptides.

[0051] Figure 20 shows the structure of a star-shaped block copolymer containing G2-PLL, G3-PLL, G2-PLA, G3-PLA, G3-PLL-MA, G3-PLL-VA, and G3-PLL-CA.

[0052] Figures 21A to 21D show the cytotoxic activity of the block copolymers. Figure 21A is a bar graph showing the survival rate (%) of A549 cells treated with the copolymers (G2-PLL10 (with SEQ ID NO: 13 branch), G2-PLA10, G3-PLL9 (with SEQ ID NO: 15 branch), and G3-PLA9). Figure 21B is a bar graph showing the survival rate (%) of H1299 cells treated with the copolymers (G2-PLL10 (with SEQ ID NO: 13 branch), G2-PLA10, G3-PLL9 (with SEQ ID NO: 15 branch), and G3-PLA9). Figure 21C is a bar graph showing the survival rate (%) of A549 cells treated with the copolymers (G3-PLL9-CA0.3, G3-PLL9-VA0.25, and G3-PLL9-MA0.2). Figure 21D is a bar graph showing the survival rate (%) of H1299 cells treated with the copolymers (G3-PLL9-CA0.3, G3-PLL9-VA0.25, and G3-PLL9-MA0.2). Figure 21E is a bar graph showing the survival rate (%) of 3T3 cells treated with the copolymer (G3-PLL10-PLF5 with SEQ ID NO:17 branch). Figure 21F is a bar graph showing the survival rate (%) of RAW cells treated with the copolymer (G3-PLL10-PLF5 with SEQ ID NO:17 branch).

[0053] Figures 22A and 22B show the transfection efficiency of the copolypeptides. Figure 22A is a series of graphs showing the transfection efficiency of various copolypeptides. Figure 22B is a bar chart showing the transfection efficiency (%) of various copolypeptides.

[0054] Figure 23A is a bar graph showing the survival rate (%) of H1299 cells treated with the dendritic polymer / pDNA complex. Figure 23B is a bar graph showing the survival rate (%) of 293T cells treated with the dendritic polymer / pDNA complex.

[0055] Figure 24 shows the results of cancer cell scratch healing assays after treatment with various copolypeptides (G3-PLL9 (with SEQ ID NO: 15 branch), G3-PLA9, G3-PLL9-CA0.3, G3-PLL9-VA0.25, and G3-PLL9-MA0.2). The figures are visualized at 100x magnification at 0 and 24 hours. Copolypeptide treatment inhibited the migration ability of cancer cells.

[0056] Figure 25A is a bar graph showing the wound extent response (%) after treatment with the copolymer peptides (G3-PLL9 (with SEQ ID NO:15 branch), G3-PLA9, G3-PLL9-CA0.3, G3-PLL9-VA0.25, and G3-PLL9-MA0.2). Figure 25B is a bar graph showing the cell migration response (%) after treatment with the copolymer peptides (G3-PLL9 (with SEQ ID NO:15 branch), G3-PLA9, G3-PLL9-CA0.3, G3-PLL9-VA0.25, and G3-PLL9-MA0.2).

[0057] Figure 26 is a set of graphs showing the apoptosis response of H1299 cancer cells treated with copolypeptides (G3-PLL9 (with SEQ ID NO:15 branch), G3-PLA10, G3-PLL9-CA0.3, G3-PLL9-VA0.25, and G3-PLL9-MA0.2) at 0', 10', 40', and 50' (min).

[0058] Figure 27 is a set of graphs showing the visual apoptosis response of 293T, H1299, and A549 cancer cells treated with G3-PLL9-VA0.25 at 0', 10', 40', and 50'.

[0059] Figure 28 shows a set of immunofluorescence staining images of juxta-apoptotic H1299 cancer cells treated with G3-PLL9-VA 0.25 at 0 μM, 0.05 μM, 0.1 μM, and 0.2 μM. Juxta-apoptotic cancer cells were stained with DAPI and Nile red.

[0060] Figure 29 shows a group of immunofluorescence staining images of juxta-apoptotic A549 cancer cells treated with G3-PLL9-VA 0.25 at 0 μM, 0.05 μM, 0.1 μM, and 0.2 μM. Juxta-apoptotic cancer cells were stained with DAPI and Nile Red. Implementation

[0061] The following embodiments are provided to illustrate the present invention in detail. Those skilled in the art can easily understand the advantages and effects of the present invention after reading this document, and it can also be implemented or applied in other different embodiments. Therefore, the following embodiments can be modified and / or altered to implement the present invention without departing from its different embodiments and scope of application, and any element or method within the scope of the present invention disclosed herein can be combined with any other element or method disclosed in any embodiment of the present invention.

[0062] As used herein, the singular forms “a,” “an,” and “the” include a plural of indicators unless explicitly and definitively limited to one indicator. Unless the context clearly indicates otherwise, the term “or” may be used interchangeably with the term “and / or.”

[0063] The term "short peptide" is used herein to refer to a series of amino acid residues, typically linked together by peptide bonds between an alpha-amino group and the carbonyl group of an adjacent amino acid. The peptide is preferably 10 or fewer amino acids long, and 2 or more amino acids long, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids long.

[0064] The terms "peptide," "polypeptide," and "copolypeptide" are used herein to refer to a series of amino acid residues having more amino acid residues than a "short peptide," such as 10 to 40 amino acid residues, 10 to 30 amino acid residues, or 10 to 25 amino acid residues, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid residues. "Copolypeptide" refers to a long amino acid sequence comprising two or more types of amino acids.

[0065] The peptides, polypeptides, or copolypeptides (hereinafter referred to as peptides) of this invention may comprise 20 standard α-amino acids (i.e., natural amino acids) used for cellular protein synthesis, as well as non-natural amino acids (which may be naturally occurring but not used for cellular protein synthesis, such as ornithine, citrulline, and sarcosine, or may be chemically synthesized), amino acid analogs, and peptidomimetic compounds. The amino acid may be a D- or L-optical isomer. The peptide may be formed by a condensation or coupling reaction between the α-carbon carboxyl group of one amino acid and the amino group of another amino acid. The terminal amino acid (amino terminus) at one end of the chain thus has a free amino group, while the terminal amino acid (carboxyl terminus) at the other end of the chain has a free carboxyl group. Alternatively, the peptide may be a non-linear, branched peptide or a cyclic peptide. Furthermore, the peptide may be modified or protected with various functional groups or protecting groups as needed, including at the amino and / or carboxyl termini.

[0066] The amino acid abbreviations in this peptide are as follows: phenylalanine is Phe or F; leucine is Leu or L; isoleucine is Ile or I; methionine is Met or M; valine is Val or V; serine is Ser or S; proline is Pro or P; threonine is Thr or T; alanine is Ala or A; tyrosine is Tyr or Y; histidine is His or H; glutamic acid is Gln or Q; aspartic acid is Asn or N; lysine is Lys or K; aspartic acid is Asp or D; glutamic acid is Glu or E; cysteine ​​is Cys or C; tryptophan is Trp or W; arginine is Arg or R; and glycine is Gly or G.

[0067] The peptide itself has therapeutic efficacy and can be used as a drug or therapeutic agent, or as an active ingredient in a pharmaceutical composition. Alternatively, the peptide can serve as a carrier, vehicle, or vector for other drugs or therapeutic agents. Other drugs or therapeutic agents may be anticancer agents, genes, or combinations thereof, such as cisplatin, oxaliplatin, nedaplatin, and / or lobaplatin.

[0068] Figure 1 illustrates the interaction of the peptide, specifically the block copolymer comprising a first positively charged peptide fragment and a second hydrophobic peptide fragment (e.g., Lys5BnCys5 (SEQ ID NO:2) and Lys5Cys5 (SEQ ID NO:1)), with target cells such as cancer cells (e.g., lung cancer cells). In at least one embodiment disclosed herein, the peptide disrupts the integrity of the target cell membrane and / or interrupts the expression or embedding of epithelial cadherin on the target cell membrane, particularly interfering with the assembly of epithelial cadherin and calcium ions to form intact epithelial cadherin through its coil-and-fold structure, thereby inhibiting the migration and attachment of the target cell and promoting its anoikis. Furthermore, apoptosis protease 3, poly(ADP-ribose) polymerase (PARP), and chromatin condensation in the target cell are triggered and activated to promote its apoptosis. The peptides disclosed herein can achieve therapeutic effects based on this theory (i.e., simultaneously through intracellular and extracellular signaling pathways), or it is believed that the therapeutic effects are not bound by this theory. In Figure 1, although fragments composed of L-lysine and fragments composed of L-cysteine ​​or S-benzyl-L-cysteine ​​are used to form coiled and β-plate shapes, respectively, other amino acids can also be used.

[0069] The peptide may contain or be modified to contain functional groups, which may attach directly to the water-soluble polymer or through spacer moieties or linkers. Functional groups include, but are not limited to, the N-terminus of the peptide, the C-terminus of the peptide, and any functional groups on the amino acid side chains of the peptide. Such amino acids include lysine, cysteine, histidine, aspartic acid, glutamic acid, tyrosine, arginine, serine, methionine, and threonine.

[0070] The terms “object,” “individual,” or “patient” are used interchangeably throughout this document and refer to vertebrates, preferably mammals. Mammals include, but are not limited to, rodents, great apes, humans, farm animals, sport animals, and pets.

[0071] "As needed" or "as needed" means that the situation described below may or may not occur, and therefore the description includes instances where the situation occurs and instances where it does not occur.

[0072] As used herein when referring to index values, the term "about" means a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% of that value. Differences in this value can occur, for example, due to experimental errors, errors common in measurement or processing procedures when preparing compounds, compositions, concentrates, or formulations, differences in the purity of the sources, preparations, or starting materials used in this invention, or similar considerations.

[0073] As used herein, the terms "comprise / comprising," "include / including," "have / having," "contain / containing," and any other variations thereof are intended to cover non-exclusive inclusion. For example, when describing an object as "comprising" a limitation, unless otherwise specified, it may additionally include other components, elements, elements, structures, regions, parts, devices, systems, steps, or connections, and should not exclude other limitations.

[0074] Short peptides include, but are not limited to, fragments, variants, and derivatives of the peptide. The short peptide also includes any and all combinations of these modifications. In non-limiting examples, a short peptide may be a fragment of a peptide as disclosed herein, or even a fragment having one or more amino acid substitutions. Therefore, it should be understood that any reference to a short peptide derived from a peptide is not limited to having only that particular modification, but includes that particular modification and any other modifications as desired.

[0075] The short peptide may arise from natural processes (such as processing and other post-translational modifications) or may be prepared by chemical modification techniques. Such modifications are well known to those of ordinary skill in the art.

[0076] The short peptide may exhibit a single or multiple variations relative to the parent peptide. When multiple variations are present, these variations may be of the same or different types.

[0077] Furthermore, modifications can occur at any position on the peptide, including the peptide backbone, amino acid side chains, and N or C terminus.

[0078] The "effective amount" of a drug or therapeutic agent is any amount of drug that, when used alone or in combination with other therapeutic agents, promotes disease remission by protecting a subject against disease onset, or by reducing the severity of disease symptoms, by increasing the frequency and duration of asymptomatic periods of disease, or by preventing injury or disability caused by disease suffering. The ability of a therapeutic agent to promote disease remission can be assessed using a variety of methods known to those of ordinary skill in the art, such as in human subjects during clinical trials, in animal model systems predicting efficacy in humans, or by determining the activity of the agent in in vitro assays.

[0079] As used herein, the term "anticancer activity" refers to a peptide capable of altering the function or metabolism of target cancer cells (e.g., affecting reproduction, growth, toxin production, subsistence, etc., but not limited to these). In one embodiment, anticancer activity refers to the inhibition of cancer cell growth. Moreover, in a specific embodiment, anticancer activity refers to the peptide's ability to kill at least one type of cancer cell.

[0080] As used herein, the term "wound healing" in this disclosure can refer to a continuous, dynamic, and complex process, which may include, but is not limited to, cell proliferation and cell migration. In one embodiment, the phrase "promoting wound healing" used in this disclosure may also refer to "enhancing cell proliferation" or "enhancing cell migration," but is not limited thereto.

[0081] The wound-healing effect of the pharmaceutical ingredients disclosed herein may also refer to effects such as "enhancing cell proliferation" or "enhancing cell migration," but it is not limited to these.

[0082] The pharmaceutical ingredients disclosed herein can be administered intravenously, intra-arterially, intraperitoneally, intramuscularly, intradermally, intratumorally, orally, transdermally, transnasally, transorally, transrectally, transvaginally, by inhalation, or topically.

[0083] The pharmaceutical composition disclosed herein can be applied to plants or animals. The aforementioned animals may include fish, birds, mammals, etc., but are not limited to these. Examples of mammals may include, but are not limited to, cats, dogs, cattle, horses, pigs, humans, etc. In one embodiment, the pharmaceutical composition of the present invention can be applied to humans.

[0084] The disclosed copolypeptide can be synthesized by ring-opening polymerization (ROP). For example, an amino acid suitable for synthesizing a fragment with the desired properties (i.e., positively charged or hydrophobic) is selected and reacted to form an N-carboxyanhydride (NCA). Depending on the need, some amino acids require an additional side-chain protection step (e.g., using a benzyloxycarbonyl (Z-group)) and this step is performed before the NCA formation step. ROP is then performed with an amine initiator and NCA to form the desired copolypeptide. If the side-chain protection step has been performed first, an additional side-chain deprotection step is required after the copolypeptide formation.

[0085] In one embodiment, L-lysine (suitable for synthesizing positively charged fragments) and S-benzyl-L-cysteine ​​(suitable for synthesizing hydrophobic fragments) are selected, and L-lysine is reacted with benzyl chloroformate to form Z-Lys (i.e., L-lysine protected by a Z-group). Subsequently, Z-Lys and BnCys react to form Z-Lys-NCA and BnCys-NCA, respectively. Z-Lys-NCA and BnCys-NCA are subjected to ROP using hexylamine as an initiator to synthesize the Z-LysmBnCysn copolypeptide, followed by deprotection of the Z-group to obtain the LysmBnCysn copolypeptide. The ranges of m and n are from 1 to 50, from 1 to 40, from 1 to 30, or from 1 to 20.

[0086] In one embodiment, a LysmCysn copolypeptide is derived from a Z-LysmBnCysn copolypeptide or a LysmBnCysn copolypeptide. Here, the Z-LysmBnCysn copolypeptide undergoes a deprotection step to form LysmBnCysn, and further reacts with a reagent capable of removing the S-benzyl group on the BnCys fragment to form LysmCysn.

[0087] In one embodiment, LysmBnCysn is Lys5BnCys5 (SEQ ID NO:2) to Ly20BnCys5 (SEQ ID NO:21), such as Lys5BnCys5 (SEQ ID NO:2), Ly10BnCys5 (SEQ ID NO:5), Lys15BnCys5 (SEQ ID NO:3), and Ly20BnCys5 (SEQ ID NO:21). In another embodiment, LysmCysn is Lys5Cys5 (SEQ ID NO:1) to Ly20Cys5, such as Lys5Cys5 (SEQ ID NO:1), Ly10Cys5 (SEQ ID NO:19), Lys15Cys5 (SEQ ID NO:20), and Ly20Cys5 (SEQ ID NO:22).

[0088] The following examples further illustrate exemplary embodiments of the present invention and should not be construed as limiting the scope of the invention.

[0089] [Characteristics of copolypeptides]

[0090] The chain lengths of the Z-Lysm, Z-LysmBnCysn, and LysmBnCysn segments disclosed herein are derived from the integral ratio of the protons on the initiator (CH3CH2-), the protons on Z-Lys (-(CH2)3CH22-), and the protons on the methyl group (-CH2SCH2- or -CH2SCH2-) on BnCys, as shown in the figure. As shown in [10A to 10C]. The integral ratio of the initiator, Z-Lys, and BnCys disclosed herein is calculated to be 1:5:5, consistent with the feed molar ratio (1:5:5). Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis showed that the number-average molecular weight (Mn) of the derivatized peptide with the side chain protected was 2300 g mol⁻¹, and it had a low molecular weight distribution (Mw / Mn = 1.08), comparable to the theoretical value (2375 g mol⁻¹). Lys5BnCys5 (SEQ ID NO:2) was obtained by removing only the Z group from the Z-Lys fragment of Z-Lys5BnCys5 using HBr. As shown in Figures 10C and 11A, 1H NMR analysis of Lys5BnCys5 (SEQ ID NO:2) confirmed that the benzyl group on the BnCys fragment was intact.

[0091] The Z and benzyl groups of the Z-Lys and BnCys fragments of Lys5BnCys5 (SEQ ID NO:2) were removed using HBr and trimethylsilyl iodide (TMSI, Alfa Aesar), respectively, to obtain Lys5Cys5 (SEQ ID NO:1). 1H NMR analysis of Lys5Cys5 (SEQ ID NO:1) revealed that the percentages of residual Z and benzyl groups were less than 12% (Figure 11B), indicating the removal of protecting groups from the Z-Lys and BnCys fragments.

[0092] The chain conformations of the dicopolymers Lys5BnCys5 (SEQ ID NO:2) and Lys5Cys5 (SEQ ID NO:1) were characterized by CD and FTIR analysis under neutral conditions. The percentage of peptide chain conformations was calculated by fitting the CD spectra using BestSel software (Beta Structure Selection). The dicopolymers exhibited a mixed random coil and β-lamella / turn conformation (Figure 12A and Table 1), consistent with the characteristic peaks of β-lamella, random coil, and β-turn at approximately 1626, 1651, and 1677 cm⁻¹, respectively, as shown by FTIR analysis (Figure 12B).

[0093] Table 1. Percentage of various secondary configurations used in this copolymer peptide.

[0094] The Cys5 and BnCys5 fragments form an intermolecular β-lamella / turn through hydrophobic and hydrogen bonding interactions. Furthermore, the percentage of β-lamella / turn configuration used in the disclosed copolymer is much higher than that of lamellar portions such as BnCysn or Cysn fragments. The percentage of β-lamella / turn configuration used in Lys5BnCys5 (SEQ ID NO:2) may be slightly higher than that used in Lys5Cys5 (SEQ ID NO:1) due to the presence of the benzyl group.

[0095] In this embodiment, the coiled poly(L-lysine) is linked to a hydrophobic lamellar peptide fragment, causing the copolymer to tend to form a bilayer assembly in aqueous solution. Figure 13 shows the SAXS plot of the copolymer solution, revealing the formation of the bilayer assembly, with I(q) at the low scattering vector (q). The scattering intensity (I(q)) of the q-2 characteristic was used to demonstrate this. The critical aggregation concentrations (cac) of Lys5BnCys5 (SEQ ID NO:2) and Lys5Cys5 (SEQ ID NO:1) were determined to be approximately 0.035 and 0.05 mg mL⁻¹ (16.63 and 30.21 μM), respectively (Figures 14A and 14B). The results showed that once the peptide concentration was above its cac value, the di-copolymer could self-assemble to form a bilayer, and Lys5BnCys5 (SEQ ID NO:2) exhibited better folding ability than Lys5Cys5 (SEQ ID NO:1), which was evidenced by the lower cac value of Lys5BnCys5 (SEQ ID NO:2) than Lys5Cys5 (SEQ ID NO:1). When forming a bilayer assembly, the confined Lys5 segment can undergo a curl-fold / turn configuration change, resulting in a high percentage of β-fold / turn configurations.

[0096] [Example 1: Lys] [5] [BnCys] [5] [(SEQ ID NO:2) and Lys] [5] [Cys] [5] [(SEQ ID NO:1) Enhances cytotoxicity and hemolytic activity]

[0097] To determine whether Lys5BnCys5 (SEQ ID NO:2) could enhance cytotoxicity in the cancer cells disclosed herein, A549 and H1299 cancer cells and BEAS-2B normal cells were treated with Lys5Cys5 (SEQ ID NO:1) or Lys5BnCys5 (SEQ ID NO:2) for 24 hours. As shown in Figures 2A and 2B, the two cancer cells (A549 and H129) treated with Lys5BnCys5 (SEQ ID NO:2) exhibited lower IC50 values ​​than those treated with Lys5Cys5 (SEQ ID NO:1) (Table 2).

[0098] Table 2. IC50 of copolypeptide treatment for cancer cells and normal cells.

[0099] Furthermore, after treatment with this dimeric peptide, normal cells (BEAS-2B) exhibited lower cytotoxicity than any type of cancer cell (Figure 2C). This phenomenon can also be observed with treatment using peptide fibril assemblies.

[0100] Furthermore, the addition of low-dose cisplatin (CP) enhanced peptide-induced cytotoxicity in these cells (Figures 2A to 2C). To further confirm the cytotoxicity to suspension cells and the potential for in vivo application, human erythrocytes (RBCs) were co-cultured with the peptides for 1 hour. As shown in Figure 2D, RBCs treated with Lys5BnCys5 (SEQ ID NO:2) exhibited greater hemolytic activity than those treated with Lys5Cys5 (SEQ ID NO:1). Overall, these results reveal that both Lys5Cys5 (SEQ ID NO:1) and Lys5BnCys5 (SEQ ID NO:2) exhibit cytotoxicity in attached and suspension cells, with Lys5BnCys5 (SEQ ID NO:2) exhibiting more enhanced cytotoxicity.

[0101] [Example 2: Lys] [5] [BnCys] [5] [(SEQ ID NO:2) and Lys] [5] [Cys] [5] [(SEQ ID NO:1) Inhibits lung cancer cell migration and attachment]

[0102] To investigate whether the copolypeptide could affect cell migration, a wound healing assay was performed. After the incision was made, H1299 cells were treated with either the copolypeptide or, additionally, with cisplatin (CP) for 24 hours. As shown in Figures 3A and 3B, both copolypeptides inhibited cell migration, and the additional low-dose cisplatin (5 μM) enhanced this effect. Furthermore, at the same concentration, treatment with Lys5BnCys5 (SEQ ID NO:2) showed a larger wound healing extent than treatment with Lys5Cys5 (SEQ ID NO:1).

[0103] To further confirm whether the benzyl group on Lys5BnCys5 (SEQ ID NO:2) participates in regulating cell migration, the cell membrane was stained with 1,1'-diethyl-4,4'-carbonylcyanine iodide. Like most lipophilic carbonylcyanine staining agents, 1,1'-diethyl-4,4'-carbonylcyanine iodide staining is used for phospholipid bilayer labeling to track membrane changes. As shown in Figure 15, A549 and H1299 cells treated with Lys5BnCys5 (SEQ ID NO:2) for 6 hours showed high staining with 1,1'-diethyl-4,4'-carbonylcyanine iodide, but those treated with Lys5Cys5 (SEQ ID NO:1) did not. To further confirm whether Lys5BnCys5 (SEQ ID NO:2) can interfere with cell adhesion, the cell adhesion rate was determined after treatment with this copolymer peptide. As shown in Figure 3C, Lys5BnCys5 (SEQ ID NO:2) significantly affected cell adhesion compared to Lys5Cys5 (SEQ ID NO:1), and the addition of low-dose cisplatin also promoted this phenomenon (Figure 3C). These results suggest that Lys5BnCys5 (SEQ ID NO:2) further integrates into the membrane, causing the membrane to lose its integrity, as observed in 1,1'-diethyl-4,4'-carbonylcyanine staining. The loss of integrity caused by Lys5BnCys5 (SEQ ID NO:2) reduces cell migration. Therefore, the benzyl group on Lys5BnCys5 (SEQ ID NO:2) appears to be involved in the inhibition of cell migration.

[0104] [Example 3: Lys] [5] [BnCys] [5] [(SEQ ID NO:2) and Lys] [5] [Cys] [5] [(SEQ ID NO:1) Affects epithelial cadherin expression and induces apoptosis signals]

[0105] In this disclosure, epithelial cadherin is one of the key molecules involved in cell-cell adhesion in cancer progression. To confirm whether Lys5BnCys5 (SEQ ID NO:2) and Lys5Cys5 (SEQ ID NO:1) can affect cell adhesion and apoptosis signaling, the performance of epithelial cadherin, apoptosis protease 3, and PARP in H1299 was evaluated after treatment with the copolymer peptide for 24 hours. As shown in Figure 4A, Lys5BnCys5 (SEQ ID NO:2) and Lys5Cys5 (SEQ ID NO:1) significantly reduced the performance of epithelial cadherin and triggered the performance of apoptosis protease 3 and PARP, with Lys5BnCys5 (SEQ ID NO:2) showing a stronger effect than Lys5Cys5 (SEQ ID NO:1).

[0106] The location of epithelial cadherin in stained cells was also assessed to evaluate cell-cell interactions after peptide treatment. Cells treated with Lys5BnCys5 (SEQ ID NO:2) became more rounded (i.e., segregated) than those treated with Lys5Cys5 (SEQ ID NO:1), which was observed in the brown-stained membrane epithelial cadherin (…). [Figure 16]). The downregulation of epithelial cadherin and the upregulation of apoptosis signals were both enhanced by the addition of the copolymer and low-dose cisplatin (5 μM).

[0107] The DNA condensation step, a signal of apoptosis, was also observed to begin in Figure 4B, consistent with the results of Western ink dot assay (Figure 4A). Treatment with Lys5BnCys5 (SEQ ID NO:2) also resulted in flatter cells, increased leakage of intracellular content, and significant loss of original saturation (Figure 4B, visible light image). In this Lys5BnCys5 (SEQ ID NO:2) treated group, the cell membrane showed tearing and clustering similar to those observed with Nile Red staining (Figure 4B). These results are consistent with migration assays, supporting the effects of this benzyl-enhancing copolypeptide on cell-cell adhesion, cell migration, and apoptosis. Therefore, Lys5BnCys5 (SEQ ID NO:2) and Lys5Cys5 (SEQ ID NO:1) are considered effective apoptosis inducers and migration inhibitors, with Lys5BnCys5 being the more effective one.

[0108] Those skilled in the art will understand that, in certain instances, epithelial cadherins play a complex role in the process of promoting and inhibiting tumor metastasis at different stages. However, molecules that inhibit cadherin-mediated survival in metastatic cancers have been suggested as potential therapeutic agents. This disclosure provides that the copolymeric peptide treatment inhibits cancer cell migration and attachment (Figure 3), and also provides information on the interaction of the copolymeric peptide with the cell membrane and its influence on normal epithelial cadherin expression through electrostatic, hydrophobic, and hydrogen bonding interactions (Figures 4, 15, and 16). It is believed that the cationic Lys fragment and the bulky BnCys or Cys fragment described herein bind to negatively charged cadherin molecules and act as spacers to prevent cadherin folding, respectively. Moreover, the binding of the peptide to the cell membrane or epithelial cadherin molecules significantly alters the peptide's amphiphilicity, facilitating peptide chain folding onto the cell membrane or between epithelial cadherin molecules. In this disclosure, it is believed that the influence of the aforementioned copolypeptide on the epithelial cadherin of the target cells and the disruption of the integrity of the target cell membrane caused by the copolypeptide embedding into the lipid bilayer inhibit the migration and attachment of the target cells and trigger and activate apoptosis protease 3 and PARP in the target cells, thereby inducing apoptosis (Figures 9 and 1).

[0109] [Example 4: Lys] [5] [BnCys] [5] [(SEQ ID NO:2) exhibits anti-cancer effects in mouse models with the same gene]

[0110] To test the anticancer effect of Lys5BnCys5 (SEQ ID NO:2), an in vivo syngeneic lung cancer model was used. Briefly, LL2 cells transfected with the luciferase reporter gene were subcutaneously inoculated into C57BL / 6 mice. Upon activation of the luciferase gene, compared to the saline control group, fluorescence results indicated lower bioluminescence in all experimental copolypeptide groups (Figs. 5A and 5B), revealing that exposure to Lys5BnCys5 (SEQ ID NO:2) inhibited tumor growth. Cisplatin is commonly used to treat lung cancer, but has been reported to have side effects such as weight loss (an important indicator of cachexia), forcing patients to discontinue treatment. In this disclosed model, the combination therapy of Lys5BnCys5 (SEQ ID NO:2) and cisplatin demonstrated stronger tumor growth inhibition than treatment with the copolypeptide alone (Figs. 5C and 5D). As expected, all groups treated with cisplatin showed weight loss (Fig. 5E), but mice treated with the peptide alone did not show any significant weight loss, while the group treated with a combination of the peptide and cisplatin showed comparable weight loss to those treated with cisplatin alone (Fig. 5E). Notably, mice treated with the peptide showed significantly longer survival times than the control group (Fig. 5F). Based on these results, the efficacy against tumor metastasis was tested by investigating the ability of Lys5BnCys5 (SEQ ID NO:2) to inhibit lung metastatic nodules. Once the saline control group with tumors reached a volume of 1800 to 2000 mm3 (32 days) after treatment, the tumors were removed (Figs. 6A and 6B). The peptide-treated tumors showed significantly lower numbers and sizes of metastatic nodules (Figs. 6C and 6D). These results reveal that Lys5BnCys5 (SEQ ID NO:2) can effectively inhibit tumor growth and prolong the survival time of tumor-bearing mice. Lys5BnCys5 (SEQ ID NO:2) combined with cisplatin further improves efficacy.

[0111] Based on these results, this disclosure found that the reduction in epithelial cadherin expression (as seen in immunoblotting) is achieved through Lys5BnCys5 (SEQ ID NO:2) influencing protein folding, rather than through gene regulation. As shown in Figures 6C and 6D, these in vivo anti-metastatic results are consistent with the syngeneic lung cancer model disclosed in this disclosure. Furthermore, Lys5BnCys5 (SEQ ID NO:2) treatment induced the apoptosis protease 3-dependent apoptosis pathway but did not induce adverse reactions such as weight loss (Figure 5E). As shown in Figures 6A to 6D and Figures 7A to 7D, Lys5BnCys5 (SEQ ID NO:2) treatment significantly reduced tumor growth and prolonged survival in tumor-bearing mice (Figure 5F), suggesting that Lys5BnCys5 (SEQ ID NO:2) treatment could serve as an anti-cancer peptide.

[0112] [Example 5: Lys] [5] [BnCys] [5] [(SEQ ID NO:2) Treatment increases the relative proportion of Ekmanophilus in the gut]

[0113] Those with ordinary knowledge in the art will understand that in some cases, lung cancer patients with high microbial diversity have prolonged progression-free survival (PFS) compared to those with low microbial diversity. To explore whether the composition of the gut microbiota is altered by Lys5BnCys5 (SEQ ID NO:2), and whether this treatment-mediated microbial alteration may be related to its effect on tumor growth, the proportion of 16S rDNA amplicones in each treatment group was analyzed. Although the Lys5BnCys5 (SEQ ID NO:2) treatment group showed lower levels of α-diversity based on both abundance and evenness indices (Fig. 7A, P = 0.027), co-treatment with cisplatin significantly increased gut microbial α-diversity (Fig. 7A, P = 0.029), suggesting that Lys5BnCys5 (SEQ ID NO:2) may provide some survival benefit to patients treated with cisplatin as first-line therapy. Further analysis of β-diversity was performed using principal coordinate analysis (PCoA) D_0.5 UniFrac. PERMANOVA showed significant differences in overall microbial composition among the groups (Fig. 7B, P < 0.001). The top ten most abundant gut bacteria in the genus stratum are shown in the OUT table (Fig. 7C). Interestingly, compared to pre-treatment and the saline group (data not shown, P < 0.01 and < 0.05, respectively), treatment with Lys5BnCys5 (SEQ ID NO:2) reduced the number of Ruminococcus spp. species. Notably, the anti-PD1 non-responder group exhibited a predominance of Ruminococcus spp. species.

[0114] Furthermore, heatmaps show the abundance of gut microbiota in each sample across the four subgroups in different clusters (Figs. 8A to 8D). At the phylum level, Lys5BnCys5 (SEQ ID NO:2) treatment significantly increased Verrucomicrobia compared to the pre-treatment and saline groups (Figs. 8A and 8B, P <0.05 and <0.05, respectively). Simultaneously, compared to the pre-treatment and saline groups (Figs. 8C and 8D, P <0.05 and <0.05, respectively), the number of *Akk* (the only species in Verrucomicrobia) increased after Lys5BnCys5 (SEQ ID NO:2) treatment, suggesting that Akk proliferation is one of the potential mechanisms by which Lys5BnCys5 (SEQ ID NO:2) influences tumor growth. For example, Akk has been found to enhance the anticancer effect of cisplatin in Lewis lung cancer mice.

[0115] Those with ordinary knowledge in the field will understand that, in some instances, Akk is one of the most dominant bacteria in the mucus layer of the gut, constituting 1 to 4% of all bacterial cells in the feces of healthy adults, and also understands that Akk may have beneficial effects on its host. For example, differential expression of tumor-related genes and the prevention of tumor microenvironment by increasing Akk to alter the gut microbiome in Pten-deficient mice. The relative abundance of Akk is also associated with immune checkpoint inhibitors (ICIs) through regulation of the PD-1 / PD-L1 axis. ICIs have attracted sustained clinical responses in a significant proportion of cancer patients, achieving significant efficacy in the treatment of advanced lung cancer.

[0116] Those skilled in the art will understand that, in certain instances, the gut microbiota participates in the body's immune regulation and optimizes the therapeutic effects of immune checkpoint inhibitors during cancer progression. Immune checkpoint blockade in cancer immunotherapy also exhibits heterogeneous therapeutic effects in different individuals, partly attributable to the gut microbiota. Akk is one of the most dominant bacteria and resides in the mucus layer of the intestine, accounting for 1 to 4% of the total bacteria in the feces of healthy adults. In this invention, Lys5BnCys5 (SEQ ID NO: 2) influences the composition of the gut microbiota during treatment. After three weeks of treatment with Lys5BnCys5 (SEQ ID NO: 2), Akk significantly increased compared to pre-treatment, saline, and Lys5BnCys5 (SEQ ID NO: 2) mixed with cisplatin groups. [Figures 8A to 8D]). The anticancer efficacy of Lys5BnCys5 (SEQ ID NO:2) disclosed herein may be enhanced by modulation of the gut microbiota.

[0117] According to the embodiments, the copolymer peptide disclosed herein not only affects the migration and attachment of target cells (such as cancer cells) but also induces apoptosis. These effects may be due to its interference with the expression and / or embedding of epithelial cadherins on the cell membrane, triggering apoptosis-related apoptosis signals such as apoptosis protein 3 and PARP, thereby causing DNA condensation. Mouse studies using the same gene disclosed herein showed that treatment with this copolymer peptide plus cisplatin effectively inhibited tumor growth and metastasis without weight loss and prolonged the survival time of tumor-bearing mice. Furthermore, the copolymer peptide therapy of the present invention increases the relative proportion of Akk in the gut microbiota. The synergistic effect of this copolymer peptide in inhibiting epithelial cadherins (cell migration / attachment) and regulating the gut microbiota demonstrates potent anticancer activity.

[0118] [Example 6: Astral copolymer peptides inhibit cancer cell migration and adhesion]

[0119] To explore more copolypeptides that may affect cell migration, a wound healing assay was performed using a procedure similar to that described above for linear (single-chain) and shorter copolypeptides (i.e., Lys5BnCys5 (SEQ ID NO:2) and Lys5Cys5 (SEQ ID NO:1)). After the incision was made, H1299 cells were treated for 24 hours with copolypeptides G3-PLL9 (with the side chain of SEQ ID NO:15), G3-PLA9, G3-PLL9-CA0.3, G3-PLL9-VA0.25, or G3-PLL9-MA0.2 (their chemical structures are shown in Figure 20). As shown in Figures 24, 25A, and 25B, both G3-PLL9 (with the side chain of SEQ ID NO: 15) and G3-PLA9 inhibited cell migration, and this effect was enhanced by modifying the poly-L-lysine side chain with low doses of cinnamaldehyde (CA, 0.3 μM), vanillin (VA, 0.25 μM), or p-methoxybenzaldehyde (MA, 0.2 μM). Furthermore, at the same concentration, treatments with G3-PLL9-VA0.25 and G3-PLL9-MA0.2 showed a larger wound healing area than those with G3-PLA9. The loss of integrity caused by G3-PLL9-VA0.25 and G3-PLL9-MA0.2 reduced cell migration. Therefore, the addition of VA or MA groups to G3-PLL9 appears to play an important role in inhibiting cell migration.

[0120] [Example 7: Enhanced cytotoxicity of linear copolypeptides against cancer cells]

[0121] To confirm the response to treatment with various linear copolypeptides, cell viability (%) was determined using a procedure similar to that described above for linear (single-chain) and shorter copolypeptides (i.e., Lys5BnCys5 (SEQ ID NO:2) and Lys5Cys5 (SEQ ID NO:1)), and the results are shown in Figure 19. Four types of linear copolypeptides with various fragment lengths were used: poly-L-lysine (PLL15, SEQ ID NO:7), poly-L-guanidinized lysine (PLGL15), poly-L-lysine-block-poly-S-benzyl-L-cysteine ​​(PLL-b-PBLC, e.g., PLL15-b-PBLC5 (SEQ ID NO:3), PLL10-b-PBLC9 (SEQ ID NO:4), PLL10-b-PBLC5 (SEQ ID NO:5), and PLL5-b-PBLC5 (SEQ ID NO:1)). NO:6), and poly-L-guanidinolysine-block-poly-S-benzyl-L-cysteine ​​(PLGL-b-PBLC, e.g., PLGL15-b-PBLC5, PLGL10-b-PBLC9, PLGL10-b-PBLC5, and PLGL5-b-PBLC5) were used to treat cancer cells (H1299) at concentrations ranging from 0 to 100 μM. The results showed that cell viability decreased with increasing linear copolypeptide concentration.

[0122] [Example 8: Astral copolypeptides enhance cytotoxicity against cancer cells]

[0123] Similar to Example 7, cell viability (%) was determined using a similar procedure as described above. In Figures 21A to 21D, star-shaped copolypeptides of 0.025, 0.05, 0.1, 0.25, 0.5, and 1 μM were used: G2-PLL10 (with the side chain of SEQ ID NO: 13), G2-PLA10, G3-PLL9 (with the side chain of SEQ ID NO: 15), G3-PLA9, G3-PLL9-CA0.3, G3-PLL9-VA0.25, or G3-PLL9-MA0.2 (Figure 20 shows their chemical structures, where G3-PLL9-CA0.3 corresponds to G3-PLL-CA with [1-x]=9 and m=0.3; G3-PLL9- VA0.25 corresponds to G3-PLL-VA with [1-x]=9 and m=0.25; G3-PLL9-MA0.2 corresponds to G3-PLL-MA with [1-x]=9 and m=0.2. Treatment of A549 and H1299 cells with these concentrations resulted in a significant decrease in cell viability with increasing concentration.

[0124] On the other hand, in Figures 21E and 21F, 0 to 200 μM of the star-shaped copolypeptide G3-PLL10-PLF5 (with the side chain of SEQ ID NO: 17) (i.e., G3-PLL with m=10 in Figure 20 and further containing the poly-L-phenylalanine fragment with n=5 as shown in Figure 17) was also used to treat 3T3 and RAW cells, and the decrease in cell viability became more pronounced with increasing concentration.

[0125] [Example 9: Astrogenic Copolymer Induces Apoptosis in Cancer Cells]

[0126] As shown in Figure 26, after treatment with G3-PLL9 (with the side chain of SEQ ID NO: 15), G3-PLA10, G3-PLL9-CA0.3, G3-PLL9-VA0.25, or G3-PLL9-MA0.2 for 50 minutes, flatter cells, leakage of cell contents, and loss of initial saturation were observed in H1299 cells. In Figures 27-29, compared to the control group (293T cells), G3-PLL9-VA0.25 treatment resulted in flatter cells, leakage of cell contents, and loss of initial saturation in H1299 and A549 cells. Specifically, as shown in Figure 27, these cell structures exhibit tearing and clustering, as observed in the figure approximately 50 minutes after G3-PLL9-VA0.25 treatment.

[0127] [Example 10: Linear and star-shaped copolymers increase transfection efficiency]

[0128] LysmBnCys5 copolymers (m=5, 10, and 15, i.e., SEQ ID NO: 2, 5, and 3) efficiently carry the p53 gene (pcDNA3 p53 WT plastids) into H1299 (p53-deficient) cells and enhance chemotherapy. The positively charged polymers and plastid DNA formed by LysmBnCys5 exhibited improved transfection efficiency, which increased with the increasing ratio of PLL to PBLC blocks. This improved transfection efficiency was primarily attributed to optimized polymer stability, determined by peptide charge density and rigidity. Specifically, the transfection efficiency using Lys15BnCys5 (SEQ ID NO: 3) as the gene vector was almost equivalent to that using lipofectamine 2000. This in vitro assay showed that the LysmBnCys5 gene vector reversed cisplatin sensitivity in p53-free (p53-null) cancer cells by increasing apoptosis signaling. Furthermore, studies on xenograft mice have revealed that LysmBnCys5 / p53 gene therapy significantly inhibits tumor growth and enhances low-dose cisplatin treatment. Moreover, treatment with the LysmBnCys5 / p53 gene vector and cisplatin prolonged the survival rate of tumor-bearing mice.

[0129] Figures 22A and 22B show the transfection efficiency of the p53 gene with various astrogenic copolymers into H1299 cells. Among the various astrogenic copolymers, G3-PLL9-VA0.25 showed the best transfection efficiency, exceeding that of lipofectamine 2000 by more than half. The cell viability (H1299 and 293T cells) in response to treatment with the astrogenic copolymer / pDNA complex is shown in Figures 23A and 23B, revealing that this treatment did not destroy normal cells (293T).

[0130] [Materials and Methods]

[0131] Synthesis of linear copolymer peptides (Lys m BnCys n and Lys m Cys n)

[0132] L-lysine reacts with benzyl chloroformate to prepare ZL-lysine (Z-Lys). Z-Lys and S-benzyl-L-cysteine ​​(BnCys) react to form N-carboxyhydric anhydrides (NCA): Z-Lys-NCA and BnCys-NCA, respectively. Using hexylamine as a starting agent, Z-LysmBnCysn copolypeptide is synthesized from Z-Lys-NCA and BnCys-NCA via ring-opening polymerization (ROP), and the LysmBnCysn copolypeptide is obtained by deprotecting the Z group (e.g., using hydrogen bromide HBr).

[0133] For the synthesis of this LysmCysn copolypeptide, the Z-LysmBnCysn copolypeptide was dissolved in trifluoroacetic acid (TFA, Sigma-Aldrich) in a round-bottom flask covered with aluminum foil to protect it from light. A 33 wt% hydrogen bromide solution (Sigma-Aldrich) was added to the solution, and the mixture was stirred for 30 minutes. Then, trimethylsilyl iodide (TMSI, Alfa Aesar) was added, and the mixture was stirred for another 2 hours. The crude product was obtained by pouring the mixture into excess diethyl ether (>99%, ECHO), centrifuging, and then dissolving it in deionized (DI) water. After adjusting the pH of the solution to neutral, the resulting solution was dialyzed against DI water using a 1 kD dialysis tube for 48 hours. Finally, the LysmCysn peptide was obtained by lyophilization.

[0134] Instrumentation and Characterization of Linear Copolymers

[0135] The peptides Lys5BnCys5 (SEQ ID NO:2) and Lys5Cys5 (SEQ ID NO:1) dissolved in DMSO-d6, and Z-Lys5 and Z-Lys5BnCys5 peptides dissolved in TFA-d1 were analyzed by BRUKER ADVANCE III HD NMR (600 MHz). The MALDI-TOF MS spectra of the side-chain protected peptides were recorded in reflectance mode on a Bruker Autoflex III TOF / TOF spectrometer. The secondary structures of the peptides were characterized by Fourier transform infrared (FTIR) and circularly polarized dichroism (CD) analyses using a Thermo Nicolet 6700 FTIR spectrometer and a JASCO J-815 spectrometer, respectively. For CD measurements, the concentration of the peptide sample in DI water was 0.1 mg mL⁻¹. SAXS patterns of peptide solutions were recorded using a Bruker NanoSTAR U diffractometer (controlled voltage 45 kV and current 650 μA) at room temperature and 4 × 10⁻¹ torr. The critical aggregation concentration (cac) of the peptides was determined by measuring the pyrene emission spectra of various peptide solutions using a Hitachi FL-4500 fluorescence spectrometer with an integration time of 1.0 sec. For these measurements, peptide solutions with concentrations ranging from 5 × 10⁻³ to 0.5 mg mL⁻¹ were prepared (Huang et al. 2011, and Chen et al. 2015).

[0136] Synthesis of star-shaped copolypeptides (3-arm or 6-arm Lys m-Cys n or Lys m-BnCys n)

[0137] Three-armed or six-armed star-shaped copolymers (e.g., three-armed or six-armed Lysm-Cysn or Lysm-BnCysn) were synthesized using their respective polyol initiators: 1,1,1-tris(hydroxymethyl)propane (THMP, 98%, Aldrich) and dipentaerythritol (DPET, 90%, Alfa Aesar), promoted by 1,1,3,3-tetramethylguanidine (TMG, 99%, Aldrich). For the synthesis of the three-armed Z-Lys15-BnCys5, Z-Lys-NCA solution (in 1.0 M anhydrous dimethylformamide (DMF)) and THMP solution (in 0.02 mM anhydrous DMF) were prepared in a glove box. Then, a TMG stock solution (in 54.5 mM DMF) was prepared and added to THMP solution to achieve a final concentration of 6.0 mM. After adding a specified amount of this solution to Z-Lys-NCA solution, the reaction mixture was stirred at 30°C under an argon atmosphere for 72 hours. A specified amount of BnCys-NCA (in 1.0 M anhydrous DMF) was added to the reaction mixture and stirred for an additional 72 hours at 30°C. The final molar ratio of THMP, Z-Lys-NCA, and BnCys-NCA was set at 1:60:15. Finally, after dialyzing the reaction mixture against methanol and deionized (DI) water for 48 and 24 hours, respectively, the reaction mixture was lyophilized to give a white solid. The typical yields of linear and star-shaped diblock copolymers are between 80% and 90%.

[0138] For the synthesis of the 3-arm Lys15BnCys5 copolypeptide (with the side chain of SEQ ID NO:3), the 3-arm Z-LysmBnCysn copolypeptide was first dissolved in trifluoroacetic acid (TFA, Sigma-Aldrich) in a round-bottom flask covered with aluminum foil to protect it from light, and then 33 wt% hydrogen bromide solution (Sigma-Aldrich) was added to the solution. The mixture was stirred for 30 minutes, then precipitated with diethyl ether (>99%, ECHO), dialyzed with DI water, and lyophilized (yield: 90 to 95%).

[0139] Characterization of star-shaped copolypeptides

[0140] Star-shaped copolypeptides were characterized by NMR, gel chromatography-light scattering (GPC-LS), and MALDI-TOF MS. NMR results were obtained using 2D 1H-13C HSQC NMR. The GPC-LS system, equipped with three Viscotek detectors and two Shodex GPC columns, was operated at 55 °C and a flow rate of 0.8 mL / min. The dissolution buffer was DMF containing 0.1 M LiBr, and polystyrene (molecular weight: 25000 g / mol) was used as the calculation standard. Samples were dissolved in DMF and passed through PTFE filter paper (0.2 μm, 13 mm, Finetech) before GPC analysis. MALDI-TOF MS spectra were recorded using a Bruker Autoflex III TOF / TOF spectrometer in reflectance mode.

[0141] Cytotoxicity and hemolytic assay

[0142] A549, BEAS-2B, and H1299 cells (5 × 10³ cells / well) were cultured in 96-well plates for 24 hours using 10% FBS DMEM as the medium to assess the cytotoxicity of the copolypeptide at various concentrations or in combination with cisplatin (5 μM). After adding CCK-8 reagent (Targetmol) dissolved in serum-free medium, the plates were incubated at 37°C for 1 hour. After transferring the medium to new 96-well plates, the absorbance of each well was measured at 450 nm using a microplate spectrophotometer (BioTek). The percentage of viable cells was calculated using the following formula:

[0143]

[0144] The hemolytic activity of the copolypeptide was assessed by processing human erythrocytes (hRBCs). hRBCs were purified by centrifugation, washing with phosphate-buffered saline (PBS; pH 7.0, 35 mM phosphate, 150 mM NaCl), and resuspending in PBS to 10% (v / v). This procedure was repeated three times. In sterile 96-well plates, 100 μL of the specified concentration of copolypeptide solution was added to 100 μL of the hRBC suspension, and the plates were incubated at 37°C for 1 hour. Subsequently, the plates were centrifuged at 1500 × g for 5 minutes. After transferring the supernatant to a new 96-well plate, the absorbance (Abs) of the supernatant at 405 nm was measured using a microplate reader to calculate heme release. Abs values ​​(Abs) were measured in PBS and 0.1% Triton X-100 to define 0% and 100% hemolysis, respectively. The percentage of hemolysis was calculated as [(Abs peptide-AbsPBS) / (Abs 0.1% Triton X-100-AbsPBS)] × 100.

[0145] Wound healing test

[0146] A wound healing assay was performed to assess the effect of the copolypeptide on the migration ability of H1266 cells. After seeding cells (1 × 10⁶ cells / well) into a 6-well plate, the monolayer of cells was scraped using a 200 μL micropipette tip, followed by washing three times with PBS. Fresh medium (10% FBS DMEM) with or without the copolypeptide (4 μM or 8 μM), and its combination with cisplatin (5 μM), were then added to the wells containing the scraped cells. After 24 hours of incubation, images were taken using an inverted microscope, and the extent of wound healing was quantified using ImageJ software. The percentage of wound healing was determined based on the total cell extent (100%).

[0147] Immunomodulator analysis

[0148] H1299 cells were treated with a copolypeptide (4 μM) for 24 hours in the presence or absence of cisplatin (5 μM), followed by extraction with RIPA Lysis and Extration Buffer (Thermo Fisher Scientific), and then separation using 8% SDS-PAGE for immunoblotting analysis. After transfer to an NC membrane (0.22 μm) and blocking with 5% nonfat dried milk, the protein was cultured with primary antibodies against anti-cadherin, anti-apoptotic protease 3, anti-poly(ADP-ribose) polymerase (PARP), and anti-β-actin, followed by culture with appropriate secondary antibodies. Visual protein bands were formed using the Immobilon Western Chemiluminescent HRP Substrate kit (Merck), and the intensity of the protein bands was quantified using ImageJ software (image processing and analysis in Java) by normalizing β-actin.

[0149] Immunohistochemical analysis

[0150] Immunohistochemical analysis was performed on H1299 cells treated with a copolypeptide (4 μM) for 20 hours in the presence or absence of cisplatin (5 μM). Treated cells were fixed with cold methanol for 10 minutes and treated with H2O2 solution (3%) in PBS for 30 minutes to block endogenous peroxidase. They were then incubated for 1 hour with Triton X-100 (0.3%) milk dilution / masking solution (10%, SeraCare) and washed twice with PBS. The samples were then incubated overnight at 4°C with mouse anti-cadherin (Proteintech) or isotype control IgG (Santa Cruz) followed by three washes with PBS. Finally, the samples were incubated for 2 hours with wasabi peroxidase (HRP) conjugated goat anti-mouse IgG (Jackson ImmunoResearch). Reactivity was visualized with 3,3'-diaminobenzidine (DAB, brown, Dako) and counterstained with hematoxylin.

[0151] Solid-phase adhesion determination

[0152] H1299 cells (2 × 10⁵ cells / well) seeded into 12-well plates were treated with or without copolypeptide (4 μM) for 6 hours for solid-phase adhesion assays. The cells were then separated from the culture plates by trypsinization, washed, and resuspended in serum-free medium. This cell suspension (1 × 10⁴ cells / well) was seeded into 96-well plates pre-coated with 2.0 μg / mL fibronectin (Sigma), and the plates were incubated at 37°C for 2 hours. After immersing the plates in PBS containing MgCl₂ (1.0 mM) to remove unattached cells, attached cells were measured using a CCK-8 assay. The Abs value of each well was measured at 450 nm using a microplate reader, corresponding to the proportion of cells attached to the coated well. Quadruple replicates were performed, and the assay was repeated three times. The percentage of attachment was calculated using the formula (Abs test / Abs control) × 100.

[0153] Mouse lung tumor model and non-invasive bioluminescent imaging based on luciferase

[0154] LL2 cells (5 × 10⁵) expressing luciferase reporter protein were subcutaneously inoculated into the backs of C57BL / 6 mice to establish a lung cancer animal model. Seven days later, either saline control or a copolypeptide was injected into the tumor to begin the timeline (day zero). Mice were injected intratumorally every two days with 2 mg / kg cisplatin, 5 mg / kg Lys5BnCys5 (SEQ ID NO:2), 10 mg / kg Lys5BnCys5, 5 mg / kg Lys5BnCys5 + 2 mg / kg cisplatin, or saline (120–150 μL per mouse based on body weight) for 24 days (n = 10–12). Tumor growth, body weight, and survival were closely monitored. Tumors were measured using calipers on two perpendicular axes, and tumor volume was calculated using the following formula: (tumor length) × (tumor width)² × 0.45. Once the tumor volume reached 2000 mm³, the mouse was sacrificed and recorded as dead. The experimental procedure complied with the regulations of Taiwan's Animal Protection Act and was approved by the Laboratory Animal Care and Use Committee of National Cheng Kung University. On day 12, mice were injected intraperitoneally with potassium D luciferin (200 μL, 10 mg / mL, Thermo Fisher Scientific), followed by anesthesia with 2% isoflurane for in vivo bioluminescence imaging. The IVIS-200 Perkin Elmer system and its integrated acquisition and analysis software (Living Image V.2.50) were used to quantify the signal. On day 32, the number of metastatic lung nodules was examined using an optical microscope.

[0155] Preparation and Characterization of Copeptide / DNA Polymers

[0156] The copolymer / DNA polymers were prepared as follows. In short, the copolymer and plasmid DNA were dissolved separately in deionized (DI) water to prepare homogeneous solutions. The final concentrations of the copolymer and plasmid DNA (or pEGFP-C1) were 5.0 μM and 4.0 μg mL⁻¹, respectively. The theoretical molecular weights used for PLL5-b-PBLC5 (SEQ ID NO:6), PLL10-b-PBLC5 (SEQ ID NO:5), and PLL15-b-PBLC5 (SEQ ID NO:3) were calculated to be 2127, 3172, and 4177 g mol⁻¹, respectively. Next, copolymers / DNA polymers with different N / P ratios were prepared by mixing specified volumes of the copolymer solution with the DNA (pcDNA3 p53 WT) solution. The mixtures were then shaken for 30 seconds. The dimensions of the copolymer / DNA polymers were characterized using a Hitachi H-7500 transmission electron microscope (TEM). Colloidal electrophoretic analysis of copolymers of peptides / DNA with various N / P ratios was performed by incubating at 25°C for 30 minutes and then performing on a 0.8% agarose gel (100V, 30 minutes).

[0157] Autonomous assembly structure of copolypeptides

[0158] The self-assembled structures of the copolypeptides were characterized by small-angle X-ray scattering (SAXS) using a NANOSTAR U SYSTEM equipped with a Vantec-2000 detector and an 1 μS X-ray source. SAXS patterns were generated by plotting the scattering intensity (I(q)) against the scattering vector (q). The copolypeptides and polymers in dissolved water were loaded into quartz capillaries for SAXS analysis. Copolypeptide solutions with a concentration of 1.0 mg / mL were prepared for SAXS analysis. For all PLL-b-PBLC copolypeptides, the N / P ratio of the polymers was 5. The voltage and current for the SAXS measurement were set to 45 kV and 0.65 mA, respectively.

[0159] Transfection efficiency of copolymer peptides / DNA polymers

[0160] 5.0 μM of the copolymer or 4.0 μL of lipofectamine 2000 (Thermo Fisher Scientific) was mixed with 4.0 μg of plasmid DNA (pEGFP-C, Clontech) in 1 mL of serum-free DMEM and incubated for 20 minutes. Once the cell concentration reached 2 × 10⁵ cells / well in 6-well plates, the medium was replaced with 1 mL of the copolymer / DNA complex (polymer) and the cells were incubated for 6 hours. Then, 2% FBS DMEM was added for another 24 hours of incubation. Next, 10% FBS DMEM was added for another 24 hours. All cells were cultured at 37°C under a humidified atmosphere of 5% CO₂. The green fluorescent protein (GFP) of transfected cells was examined under an inverted fluorescence microscope (IX71, Olympus). Quantitative assessment of transfection efficiency was also performed using flow cytometry (BD Biosciences). Transfected cells were washed with phosphate-buffered saline (PBS) and separated with 0.2 mL of 0.1% trypsin-EDTA. A total of 1 mL was collected from each well and centrifuged to remove the pellet. After removing the aqueous phase, the cells were resuspended in 1 mL of PBS. The transfection efficiency was then calculated as the percentage of cells expressing GFP, and the fluorescence of 2 × 10⁴ cells was assessed by flow cytometry.

[0161] p53 gene transfection and the anticancer effect of chemotherapy drugs

[0162] The copolypeptide (5.0 μM) was used to transfect H1299 cells cultured in 6-well plates with pcDNA3 p53 WT (1.0–4.0 μg, Addgene) plastids (2 × 10⁵ cells / well).

[0163] Bioinformatics Analysis

[0164] The original paired-end reads are cut and subjected to quality trimming (removing short reads and chimeras) before being assigned to operational taxonomic units (OTUs), which are shared with the Greengene database. 97% similarity. The original paired-end reads were also analyzed using the Ribosome Database Project (RDP) classifier in the base space. Operational taxonomic units (OTUs), α-diversity (towards agriculture index), and β-diversity (PCoA-unweighted UniFrac) were determined using the base space (Illumina), the CLC Microbial Genomics Module (Qiagen), and Graphpad Prism 7. The OTU table generated by the CLC Microbial Genomics Module was further analyzed using linear discriminant analysis of effect size (LEfSe) and a phylogenetic study reconstructing the community from recessive states (PICRUSt). LEfSe was performed using Galaxy / HutLab to identify specific microbial markers between groups, with an alpha of 0.05 for the factorial Kruskal-Wallis test / pairwise Wilcoxon test and an LDA cutoff of 2.0. PICRUSt predictions were performed using Galaxy / HutLab and based on the Kyoto Encyclopedia of Genetics and Genomes (KEGG) functional pathway database, and analyzed using Statistical Analysis of Metagenomic Profiles (STAMP) software. STAMP criteria were set, unclassified reads were removed, and p < 0.01 and effect size = 1 were used. These results identified functional pathways with significantly different abundances between groups at level 3.

[0165] Statistical analysis

[0166] Unless otherwise stated, data are reported as mean ± standard error of mean (SEM), n=3. Two-way analysis of variance and unpaired student t-tests were used to analyze the data. Insignificance (ns) indicates statistical insignificance, while p<0.05 indicates statistical significance, defined as *p<0.05, **p<0.01, and ***p<0.001.

[0167] The detailed embodiments described above are merely illustrative of preferred embodiments of this disclosure and are not intended to limit the scope of this disclosure. Therefore, all modifications and variations made by those skilled in the art should fall within the scope of the claims defined in the appended patent application.

[0168] [References]

[0169] The following references are incorporated herein by reference for providing exemplary procedures or other supplements to the details set forth herein.

[0170] YC Huang, M. Arham, JS Jan, Soft Matter 2011, 7 (8), 3975.

[0171] BY Chen, YC Huang, JS Jan, RSC Adv 2015, 5 (29), 22783.

[0172] Spatola, (1983) in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267.

[0173] TW202428299A_112100417_SEQL.xml

Claims

1. A block copolymer peptide comprising a first positively charged peptide fragment and a second hydrophobic peptide fragment, wherein, The block copolymer is a linear block copolymer, a branched block copolymer, or a star-shaped block copolymer including a polyol initiator as a core; the first positively charged peptide fragment is selected from L-lysine 5, L-lysine 9, L-lysine 10, and L-lysine 15; and the second hydrophobic peptide fragment is selected from the group consisting of S-benzyl-L-cysteine ​​5, S-benzyl-L-cysteine ​​9, S-benzyl-L-cysteine ​​10, S-benzyl-L-cysteine ​​15, L-cysteine ​​5, L-cysteine ​​9, L-cysteine ​​10, and L-cysteine ​​15.

2. The block copolymer peptide as described in claim 1, wherein, The first positively charged peptide fragment is composed of substituted or unsubstituted L-lysine.

3. The block copolymer peptide as described in claim 1, wherein, The second hydrophobic peptide fragment consists of unsubstituted L-cysteine ​​or S-benzyl-L-cysteine.

4. The block copolymer peptide as described in claim 1, wherein, At least one of the amino acids is grafted with at least one of the group consisting of p-methoxybenzaldehyde, vanillin, cinnamaldehyde, catechol, indole, phenol, and phenyl.

5. The block copolymer peptide as described in claim 1, having 3 to 24 arms.

6. The block copolymer peptide as described in claim 5, wherein, The first positively charged peptide fragment is present in at least one arm.

7. Use of an effective amount of the block copolypeptide as described in claim 1 in the preparation of a medicament for treating cancer, comprising: The drug should be administered to those who require it.

8. The use as described in claim 7, wherein, The effective dose is 5 mg / kg to 10 mg / kg of the subject's body weight.

9. The use as described in claim 7, wherein, The cancer group selected consists of lung cancer, breast cancer, ovarian cancer, brain cancer, kidney cancer, oral cancer, gastric and esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, uterine cancer, endometrial cancer, cervical cancer, stomach cancer, skin cancer, testicular cancer, prostate cancer, and thyroid cancer.

10. The use as described in claim 9, wherein, The cancer is lung cancer.

11. The use as described in claim 7, wherein, This block copolymer inhibits the migration and attachment of cancer cells and / or inhibits the expression of epithelial cadherin in cancer cells.

12. The use as described in claim 7, wherein, This block copolymer peptide serves as a carrier for anticancer agents.

13. The use as described in claim 12, wherein, The anticancer agent is cisplatin, oxaliplatin, nedaplatin, lobaplatin, p53 gene, or a combination thereof.

14. Use of an effective amount of the block copolypeptide as claimed in claim 1 in the preparation of a medicament for increasing the proportion of Akkmannii (Akk) in the gut of a desired individual, comprising: The drug was administered to the subject.

15. The use as described in claim 14, wherein, This block copolymer peptide can be applied to the subject intravenously, intra-arterially, intraperitoneally, intramuscularly, intradermally, intratumorally, orally, transdermally, transnasally, transorally, transrectally, transvaginally, by inhalation, or by local application.

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