A pancreatic enzyme-responsive bioactive peptide and nanofiber hydrogel prepared using the same and its application

Abstract

This invention belongs to the field of cancer drug technology, and particularly relates to a pancreatic enzyme-responsive bioactive peptide, a nanofiber hydrogel prepared using the same, and its applications. The trypsin-responsive bioactive peptide 1-Pept (sequence: naphthylacetic acid-phenylalanine-phenylalanine-glycine-tyrosine-lysine-cysteine-aspartic acid) can be co-assembled with the anticancer drug doxorubicin to form a 1-Pept / Dox nanofiber hydrogel. The nanofibers combined with Dox can disrupt the cytoskeleton, triggering a series of cascade events leading to cellular dysfunction, including mitochondrial damage, activation of apoptosis-related proteins, and nuclear damage, thereby synergistically causing tumor ablation. Compared with the existing anticancer drug doxorubicin alone, the 1-Pept / Dox nanofiber hydrogel exhibits significantly better anticancer effects in vitro and in vivo than pure doxorubicin, showing significant advantages in combating colon cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of cancer drugs, and in particular relates to a pancreatic enzyme-responsive bioactive peptide and a nanofiber hydrogel prepared using the same and its application. Background Art

[0002] Colorectal cancer is the fourth most common cancer worldwide, causing nearly 900,000 deaths annually. Chemotherapy, a conventional treatment for colorectal cancer, has been shown to effectively alleviate patients' disease progression. Doxorubicin, an antibiotic, is used to treat colorectal cancer. However, its nonselective distribution in the body, limited response rate, and associated toxic side effects limit its application. A growing number of studies have shown that peptides with specific sequences have multiple roles: they can be assembled into multifunctional nanocarriers for drug delivery and can also exert biological activities, including anticancer, antimicrobial, and anti-inflammatory properties. Enzyme-mediated peptide self-assembly (EISA) can regulate the in situ self-assembly or reassembly of peptides at the pericellular, intracellular, and subcellular organelle sites by overexpressing enzymes in the tumor microenvironment, thereby inducing a range of effects, such as damage to cell membrane structure, disruption of the cytoskeleton, and dysfunction of subcellular organelles. This has led to significant progress in selectively and potently killing cancer cells. By applying overexpressed enzymes to tumors, domestic and foreign research groups have developed a variety of EISA-based drug delivery systems that can not only control in situ morphological transformation but also achieve selective killing of cancer cells.

[0003] Trypsin (Tps) is a digestive enzyme produced by pancreatic acinar cells and plays a crucial role in protein digestion. A growing body of data suggests that overexpression of trypsin in colon cancer is closely associated with its progression. Therefore, the present invention utilizes trypsin, which is highly expressed in colon cancer sites, to manipulate the in situ assembly of bioactive peptides to combat colon cancer. Summary of the Invention

[0004] The purpose of the present invention is to provide a pancreatic enzyme-responsive bioactive peptide and a nanofiber hydrogel prepared using the same and its application.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for preparing a pancreatic enzyme-responsive bioactive peptide comprises the following steps: adding dichloromethane to a 2-chlorotrityl chloride resin to swell the resin, then adding condensing agents TBTU and Hobt, sequentially linking aspartic acid, cysteine, lysine, tyrosine, glycine, phenylalanine, phenylalanine, and naphthaleneacetic acid to the 2-chlorotrityl chloride resin until the sequence is complete, cutting the sequence from the resin with trifluoroacetic acid (TFA), washing it, and freeze-drying it to obtain a crude product, which is then purified by preparative HPLC to obtain a bioactive peptide 1-Pept having a sequence of: naphthaleneacetic acid-phenylalanine-phenylalanine-glycine-tyrosine-lysine-cysteine-aspartic acid, with a purity of not less than 96%.

[0007] Another object of the present invention is to provide a bioactive peptide prepared using the above preparation method.

[0008] Another object of the present invention is to use the prepared bioactive peptide and doxorubicin to co-assemble a nanofiber hydrogel. 1.0 wt% of the bioactive peptide is dissolved in a PBS solution, 0.1 eq of doxorubicin solution is added, and the mixture is allowed to stand for 2 hours to co-assemble into a red 1-Pept / Dox NFs nanofiber hydrogel. The active peptide is co-assembled with the anticancer drug doxorubicin (Dox) to form a sparse nanofiber hydrogel 1-Pept / Dox NFs, which can be transformed into a dense nanofiber structure after responding to trypsin, undergoing morphological transformation at the disease lesion site.

[0009] Another object of the present invention is to provide an application of a nanofiber hydrogel for the preparation of a drug for treating colorectal cancer. After injection of 1-Pept / Dox NFs, it can not only prolong the retention time of doxorubicin in the colon cancer site and enhance the intracellular uptake of the drug, but also exert a synergistic anti-colon cancer effect through the synergistic effect between the bioactive peptide and doxorubicin.

[0010] The present invention has the advantages that the trypsin-responsive bioactive peptide 1-Pept (whose sequence is: naphthylacetic acid-phenylalanine-phenylalanine-glycine-tyrosine-lysine-cysteine-aspartic acid, English sequence abbreviation: NapFFGYKCD, named 1-Pept) prepared by the present invention can be co-assembled with the anticancer drug doxorubicin (abbreviated as: Dox) into 1-Pept / Dox nanofiber hydrogel (denoted as 1-Pept / Dox NFs), which is used to effectively fight colorectal cancer. First, a trypsin-responsive bioactive peptide, 1-Pept, was prepared via solid-phase synthesis. It consists of the following components: i) a naphthylacetic acid-phenylalanine-phenylalanine (NapFF) sequence, which promotes peptide assembly; ii) a trypsin-responsive peptide sequence, Glycine-Tyrosine-Lysine-Cysteine ​​(GYKC), which cleaves at the carboxyl side of lysine (K); and iii) a C-terminal aspartic acid (D), which electrostatically binds to the anticancer drug doxorubicin (Dox), enabling stable loading of the drug. 1-Pept and Dox coassembled through noncovalent interactions to form injectable 1-Pept / Dox NFs. Upon contact with colorectal cancer sites, 1-Pept / Dox NFs exhibited enhanced cellular uptake via caveolae-mediated endocytosis. Subsequently, 1-Pept / Dox NFs released 1-Pept and Dox upon reaching the acidic perinuclear region. The released 1-Pept is converted into NapFFGYK (called Pept) under the catalysis of overexpressed trypsin in the cell, and then reassembles into denser Pept nanofibers (denoted as Pept NFs). Pept nanofibers combined with Dox disrupt the cytoskeleton (such as actin and tubulin), triggering a cascade of events leading to cellular dysfunction, including mitochondrial damage, activation of apoptosis-related proteins, and nuclear damage, synergistically causing tumor ablation. Compared with the existing anticancer drug doxorubicin alone, its anticancer efficacy in vitro and in vivo is significantly superior to that of pure doxorubicin, with a significant advantage in combating colon cancer.

[0011] This patent reports for the first time the bioactive peptides that respond to trypsin in colon cancer sites; the specially designed bioactive peptides can utilize non-covalent forces with chemotherapy drugs to form deformable nanostructures, which not only improves the stability of the drugs, but also enhances intracellular uptake and anti-cancer effects; by innovating the dosage forms of traditional chemotherapy drugs and developing new gel dosage forms, it is beneficial to expand the scope of drug use.

[0012] Application Prospects: Recent research indicates that the market for gels continues to experience rapid growth. This nanofiber gel not only prolongs the duration of drug action in vivo but also enhances drug uptake and anti-cancer efficacy. This not only provides a new application pathway for chemotherapy drugs against colon cancer but also offers a novel approach for developing novel localized drug delivery systems for disease treatment, demonstrating promising application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the TOF-MS image of 1-Pept prepared by the present invention, note, ESI-MS: C 54 H 62 N8O 12 S, calc.MW =1046, obsvd. [M+H] ＋ = 1047.4, [M+4H] ＋ =1050.4.

[0014] Figure 2 It is the HPLC chart of 1-Pept prepared by the present invention.

[0015] Figure 3 The 1-Pept prepared by the present invention 1 H-NMR spectrum, in the figure: (300 MHz, ([D6]DMSO).C 54 H 62 N8O 12S: 12.51 (S, 2 H), 9.16 (S, 1 H), 8.38-8.36 (d, 1 H, J = 6 HZ), 8.28-8.24 (m, 1 H), 8.14-8.13 (d, 1 H, J = 3 HZ), 8.02-7.99 (m, 1 H), 7.86-7.85 (d, 1 H, J = 3 HZ), 7.78-7.77 (d, 1 H, J = 3 HZ), 7.75-7.73 (d, 1 H, J =6 HZ), 7.59-7.57 (m, 3 H), 7.50-7.44 (m, 2 H), 7.33-7.31 (m, 1H), 7.29 (s, 1H), 7.20-7.19 (d, 4 H, J = 3 HZ), 7.16-7.13 (m, 6 H), 7.04-7.03 (d, 2 H, J =3 HZ), 6.64-6.63 (d, 2 H, J = 3HZ), 4.55-4.50 (m, 5 H), 4.31-4.27 (m, 1 H), 3.78-3.74 (m, 2 H), 3.60-3.55 (m, 2 H), 3.02-2.91 (m, 4 H), 2.74-2.71 (d, 4H, J = 9 HZ), 2.64 (s, 2 H), 2.36 (s,2 H), 1.68 (s, 1 H), 1.54-1.50 (m, 4 H), 1.26-1.23 (m, 4 H).

[0016] Figure 4 It is the HPLC elution curve of 1-Pept, 1-Pept / Tps prepared by the present invention at 4 hours and 1-Pept / Tps at 48 hours.

[0017] Figure 5 Figures depicting the physicochemical properties of 1-Pept / Dox NFs. Figure A, top, shows an optical image of 1-Pept / Dox NFs (1.0 wt% / 0.1 eq). Figure A, bottom, shows an optical image of a 1-Pept / Dox NF inscribed with "HUCM" using an injection needle. Figure B shows the CAC and MGC values ​​of 1-Pept / Dox NFs. Figure C shows a TEM micrograph of 1-Pept / Dox NFs. Scale bar, 200 nm. Figure D shows a TEM micrograph of 1-Pept / Dox NFs treated with 5.0 U / mL Tps. Scale bar, 200 nm.

[0018] Figure 6Figure 1 is a comparison of cell viability between 1-Pept / Dox NFs and free Dox solution. Figure A shows the survival rate of HT29 cells after 48 h of treatment with 1-Pept / Dox NFs or free Dox. Figure B shows the IC 50 value.

[0019] Figure 7 Figure 1 shows the mechanism of action of 1-Pept / Dox NFs and free Dox solution on colon cancer cells. Panel A shows a CLSM image of tubulin fluorescence signals in cells treated with 1-Pept / Dox NFs or Dox for 12 hours. Tubulin is stained with TubulinTracker™ Green in green, and nuclei are stained with Hoechst 33342 in blue. Scale bar: 20 μm. Panel B shows DNA damage induced by 1-Pept / Dox NFs or Dox by immunofluorescence staining. Histone H2A.X foci are shown in green, and nuclei are stained with DAPI in blue. Scale bar: 20 μm.

[0020] Figure 8 Figure 1 shows the in vitro release of 1-Pept / Dox NFs under different pH conditions. Figure A shows the cumulative release rate of Dox from 1-Pept / Dox-NFs under different pH conditions, with Dox solution as a control. Figure B shows the cumulative release rate of 1-Pept from 1-Pept / Dox NFs under different pH conditions.

[0021] Figure 9 Figures show the in vivo antitumor activity of 1-Pept / Dox NFs. Panel A shows tumor growth curves in HT29 xenograft mice treated with PBS, 1-Pept, Dox, and 1-Pept / Dox NFs. Panel B shows H&E images of tumors in each group. Scale bar, 100 μM. DETAILED DESCRIPTION

[0022] Example

[0023] A method for preparing a pancreatic enzyme-responsive bioactive peptide comprises adding 10 ml of dichloromethane to 0.2 mmol of 2-chlorotrityl chloride resin, adding 3-fold molar amounts of condensing agents TBTU and Hobt, and then adding 3-fold molar amounts of Fmoc-Asp(OtBu)-OH and reacting for 12 hours. Subsequently, under the action of 3-fold molar amounts of condensing agents TBTU and Hobt, 6-fold molar amounts of Fmoc-Cys(Mtt)-OH, Fmoc-Lys(boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, and naphthaleneacetic acid are added in sequence and connected to the 2-chlorotrityl chloride resin in sequence until the sequence is completed. The compound is cleaved from the resin with trifluoroacetic acid (TFA), washed three times with ether, and then lyophilized to obtain a crude product. The sample is purified by preparative HPLC and purified by 1 It was characterized by H-NMR, time-of-flight mass spectrometry and reversed-phase HPLC. The purity of the obtained 1-Pept was 96%. Its sequence was: naphthylacetic acid-phenylalanine-phenylalanine-glycine-tyrosine-lysine-cysteine-aspartic acid.

[0024] Another object of the present invention is to provide a bioactive peptide prepared by the above preparation method, which can be assembled into a multifunctional nanocarrier for drug delivery.

[0025] Another object of the present invention is to utilize the prepared bioactive peptide and doxorubicin to co-assemble a nanofiber hydrogel, wherein 1.0 wt % of the bioactive peptide was dissolved in a PBS solution, 0.1 eq of doxorubicin solution was added, and the mixture was allowed to stand for 2 hours to co-assemble into a red 1-Pept / Dox NFs nanofiber hydrogel.

[0026] Another object of the present invention is to provide an application of nanofiber hydrogel, which is used to prepare drugs for treating colorectal cancer and is used to effectively fight colorectal cancer.

[0027] Experiments and applications

[0028] 1.1-Synthesis and purification of Pept:

[0029] 1-Pept peptide was prepared by solid phase peptide synthesis (SPPS). First, 2-chlorotrityl chloride resin was accurately weighed and dichloromethane was added to swell the resin. Then, the amino acid sequence was sequentially connected to the 2-chlorotrityl chloride resin with the addition of condensing agents TBTU and Hobt until the sequence was completed. Then, trifluoroacetic acid TFA was used to cut the compound from the resin. After washing three times with ether, the compound was lyophilized to obtain a crude product. The sample was purified by preparative HPLC and purified by 1The product was characterized by H-NMR, time-of-flight mass spectrometry, and reversed-phase HPLC. The obtained 1-Pept had a purity of 96% and could be used for subsequent experiments.

[0030] 2. Investigation of trypsin responsiveness:

[0031] The structure of NapFFGYKCD (abbreviated as 1-Pept) contains the trypsin (Tps) response sequence GYKC. When Tps is overexpressed in colon cancer cells, GYKC is converted to NapFFGYK (abbreviated as Pept). First, trypsin (1 U / mL, Tps) was co-incubated with 1-Pept solution (1 mg / mL) to conduct enzyme catalytic kinetics experiments. At different time points, 50 μL of the solution was removed and vortexed with 150 μL of methanol. After filtration, the solution was used for liquid chromatography-mass spectrometry (HPLC-MS) analysis. Figure 4 As shown in the figure, after adding trypsin Tps to the 1-Pept solution, a new peak of Pept (NapFFGYK) was observed, and the Pept peak area increased over time. Within 72 hours, more than 90% of 1-Pept was converted to Pept after Tps treatment.

[0032] 3. Preparation and Characterization of Co-assembled 1-Pept / Dox NFs

[0033] 1-Pept (1.0 wt %) was dissolved in PBS solution, Dox solution (0.1 eq) was added, and after 2 hours, the two could be assembled into red 1-Pept / Dox NFs nanofiber hydrogel. The 1-Pept / Dox NFs nanogel fiber was placed in a syringe, and the syringe could be manipulated to write "HUCM" ( Figure 5 Figure A in the middle), proving that it has good injectable properties. A series of 1-Pept (1.0 wt %) solutions were prepared, and their minimum gelation concentration was examined within a specified time, with no flow when inverted as an indicator. The results showed that the minimum gelation concentration of 1-Pept was 0.8 wt %. Using the same method, a series of 1-Pept (1.0 wt %) solutions were prepared, and 0.1 eq of Dox solution was added. Within 2 hours, it was observed that the MGC of 1-Pept / Dox NFs was 0.5 wt %. In addition, 1-Pept / Dox NFs were prepared according to the above method and diluted into a series of concentration solutions. The critical micelle concentration (CAC) value of 1-Pept / Dox NFs was determined by dynamic light scattering. The results showed that the CAC value of 1-Pept / Dox NFs was 115 μM, which was significantly lower than the CAC value of 1-Pept NFs (185 μM) ( Figure 5TEM results show that compared with 1-Pept, 1-Pept and Dox co-assembled and formed slightly denser nanofibers, indicating that Dox played a promoting role in the assembly process ( Figure 5 After adding trypsin Tps to the 1-Pept / Dox solution, a dense network of nanofibers was observed, indicating the key role of Tps in the assembly process ( Figure 5 (Figure D in the middle).

[0034] 4. Cell Viability Assay of 1-Pept / Dox NFs

[0035] HT29 cells were seeded in 96-well plates and co-cultured with 1-Pept / Dox NFs. The ratio of 1-Pept to Dox was 10:1, the Dox concentration was set between 12.5-0.391 μM, and the 1-Pept concentration was between 125-3.91 μM. After 48 h, a fixed volume of 20 μL MTT (5 mg / mL) was added to the wells for co-incubation. After 4 h, the generated formazan was dissolved in DMSO, and the absorbance value was recorded at 490 nm using a microplate reader (Multiskan FC). The respective cell survival rates were calculated by measuring the absorbance of 1-Pept / DoxNFs and Dox pure drug solutions. IC was calculated using Origin 8.0 software. 50 The results showed that after 48 h of treatment with Dox solution and 1-Pept / Dox NFs, the cell survival rate decreased in a dose-dependent manner ( Figure 6 It is worth noting that the survival rate of HT29 cells treated with 1-Pept / Dox NFs was significantly decreased compared with the free Dox group at the same concentration. Further calculations showed that the IC 50 The value was 2.8 μM, which was 3.7 times that of Dox in 1-Pept / DoxNFs ( Figure 6 (Middle panel E). IC of 1-Pept in 1-Pept / Dox NFs 50 The IC value was 7.5 μM, which was much lower than that of 1-Pept alone. 50 The combination index (CI) of 1-Pept / Dox NFs was 0.32, indicating that 1-Pept enhanced the synergistic cytotoxicity of Dox on HT29 cells.

[0036] 5. Study on the mechanism of 1-Pept / Dox NFs cytotoxicity to colon cancer cells

[0037] To verify the perturbation effect of 1-Pept / Dox NFs on tubulin, HT29 cells were co-cultured with 1-Pept / Dox (62.5μM / 6.25μM) for 12 hours. The cells were then treated with Tubulin Tracker™ Green (1X) working solution for 30 minutes. The cells were then stained with Hoechst 33342 (10μg / mL) and observed by CLSM. Figure 7 As shown in Figure A, untreated cells exhibited well-defined microtubule structures, while disruption of microtubule structures was observed in cells treated with Dox and 1-Pept / Dox-NFs. In contrast, well-defined microtubule structures were barely observed in cells treated with 1-Pept / Dox NFs, likely due to intracellular reorganization catalyzed by overexpressed Tps.

[0038] To evaluate the effect of 1-Pept / Dox NFs on the cell nucleus, HT29 cells were seeded in a laser confocal microscopy dish and incubated with 1-Pept / Dox (62.5μM / 6.25μM) for 12 h. The cells were then treated with 4% formaldehyde and 0.1% Triton X-100. Subsequently, the cells were treated with rabbit anti-histone H2A.x polyclonal antibody for 12 h. Afterwards, the cells were incubated with AleaxFluor 488-labeled rabbit anti-rabbit IgG (H+L) for 4 h and observed using CLSM. Figure 7 As shown in Figure B, the untreated control group showed almost no green fluorescence signal, while Dox-treated cells displayed bright green fluorescence intensity, indicating DNA damage. Among all groups, 1-Pept / Dox-treated cells exhibited the strongest fluorescence signal intensity, which was 7.0 times and 2.3 times that of the blank control group and Dox group, respectively, indicating that it was significantly superior to the pure Dox solution in promoting DNA damage.

[0039] 6. In vitro release behavior of Dox and 1-Pept from 1-Pept / Dox NFs

[0040] The in vitro drug release study of 1-Pept / Dox NFs was carried out in a constant temperature oscillator (40 rpm, 37°C). First, 1-Pept / Dox NFs (200 μL, 1.0 wt% / 0.1eq) were prepared in glass vials, and 200 μL of PBS buffer (pH = 7.4, 6.5 and 5.5) were added to their surface. At specific time points, 200 μL of liquid supernatant was aspirated and mixed with an equal volume of methanol, and further analyzed by HPLC. At the same time, 200 μL of fresh PBS was added to the glass vial to maintain a fixed volume. Free Dox solution was used as a control. Figure 8As shown in Figure 1, the Dox solution exhibits a burst release pattern, with cumulative Dox release reaching 51% within 12 hours. To further explore the drug release mechanism, the Ritger-Peppas model was employed. Calculations show that the exponential factor n for the pure Dox solution is 0.35, indicating that Dox release from the free Dox solution follows a Fickian diffusion mechanism.

[0041] In all pH ranges, the cumulative release rate of 1-Pept / Dox NFs within 12 h was less than 10%, which was significantly lower than that of the free Dox solution, indicating that 1-Pept / Dox NFs has sustained release properties. Then, we studied the pH-responsive release curves under different pH conditions (7.4, 6.5 and 5.5). In neutral release medium, the cumulative release rate of Dox reached 2.7% after 24 h. In contrast, Dox was released from 1-Pept / Dox-NF at an accelerated rate in acidic dissolution medium. In media with pH 6.5 and 5.5, the cumulative release rates of 1-Pept / Dox NFs in 24 h were 5.5% and 7.2%, respectively, which were significantly higher than the release rate at pH 7.4, indicating that 1-Pept / Dox NFs have acid-responsive release properties ( Figure 8 Figure A). Further Ritger-Peppas model fitting revealed that the n values ​​of 1-Pept / Dox NFs at pH 7.4, 6.5, and 5.5 were 0.53, 0.54, and 0.58, respectively, demonstrating the non-Fickian diffusion of Dox released from 1-Pept / Dox NFs. Similarly, the cumulative release rate of 1-Pept from 1-Pept / Dox NFs at pH 7.4 was significantly slower than that at pH 6.5 and 5.5 ( Figure 8 (B). The calculated n values ​​for 1-Pept at pH 7.4, 6.5, and 5.5 were 0.54, 0.55, and 0.58, respectively, which were greater than 0.43, indicating that 1-Pept released from 1-Pept / Dox NFs undergoes non-Fickian diffusion. In summary, 1-Pept / Dox NFs exhibit acid-responsive properties, likely due to the protonation of the carboxylic acid group of 1-Pept and the weakening of the electrostatic interaction between 1-Pept and Dox.

[0042] 7. Study on the Anti-colorectal Cancer Effect of 1-Pept / Dox NFs

[0043] The HT29 tumor-bearing mouse model was established to study the anti-tumor effect of 1-Pept / Dox NFs in vivo. HT29 cell suspension (100 μL, 5.0 × 10 6cells) were mixed with an equal amount of Matrigel. The mixture was then subcutaneously injected into the right back of male BALB / C nude mice (6-8 weeks old, Jinan Pengyue Laboratory Animal Breeding Co., Ltd., China). 3 Mice were randomly divided into four groups: (1) PBS group; (2) 1-Pept NFS group; (3) free Dox solution; and (4) 1-Pept / Dox NFS group. 200 μL of the corresponding preparation was injected peritumorally on days 1, 4, 7, and 10. The doses of Dox and 1-Pept were 13 μmol / kg and 130 μmol / kg, respectively. The tumor size and body weight of the mice were measured every three days. According to the formula: V = L × W 2 Tumor size was calculated by averaging 100 μg / 2, where L and W represent the length and width of the tumor, respectively. At the end of day 21, tumor tissues were collected and stained with hematoxylin-eosin (HE) to observe changes in tumor cell morphology.

[0044] By observing the dynamic change curve of tumor volume, it can be found that the tumor in the PBS group grew fastest within the measurement time range, while subcutaneous injection of 1-Pept / Dox NFs had a significant inhibitory effect on tumor growth, indicating that 1-Pept / Dox NFs has excellent anti-tumor efficacy. In contrast, the inhibitory effect of injection of Dox or 1-Pept on tumor proliferation was weaker ( Figure 9 Figure A). Calculated tumor volume growth rates on day 21 were 5.7, 4.2, 3.3, and 1.5 for the PBS, 1-Pept, Dox, and 1-Pept / Dox-NFs groups, respectively. After treatment, tumor tissues were stained with H&E. Figure 9 As shown in Figure B, the cell density in the 1-Pept and Dox groups was slightly lower than that in the PBS group, consistent with the relative tumor volume results, further demonstrating their poor tumor inhibitory effect. In particular, tumor tissue in the 1-Pept / Dox NFs group exhibited not only the lowest cell density but also extensive necrosis, suggesting a unique role for 1-Pept in enhancing the anticancer efficacy of Dox. Therefore, 1-Pept / Dox NFs exhibited potent tumor inhibitory efficacy.

Claims

1. A method for preparing a pancreatic enzyme-responsive bioactive peptide, characterized in that: Dichloromethane is added to 2-chlorotrityl chloride resin to swell the resin, and then condensing agents TBTU and Hobt are added. Aspartic acid, cysteine, lysine, tyrosine, glycine, phenylalanine, phenylalanine, and naphthylacetic acid are sequentially connected to the 2-chlorotrityl chloride resin until the sequence is completed. The sequence is cut from the resin with trifluoroacetic acid (TFA), washed, and freeze-dried to obtain a crude product. The crude product is then purified by preparative HPLC to prepare a bioactive peptide 1-Pept, whose sequence is: naphthylacetic acid-phenylalanine-phenylalanine-glycine-tyrosine-lysine-cysteine-aspartic acid, and the purity is not less than 96%.

2. The bioactive peptide prepared by the preparation method according to claim 1.

3. A method for preparing a nanofiber hydrogel by co-assembling the bioactive peptide according to claim 2 and doxorubicin, characterized in that: 1.0 wt% of the bioactive peptide was dissolved in PBS solution, 0.1 eq of doxorubicin solution was added, and the mixture was allowed to stand for 2 hours to assemble into red 1-Pept / Dox NFs nanofiber hydrogel.

4. Use of the nanofiber hydrogel prepared by the method according to claim 3 in preparing a drug for treating colorectal cancer.

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