Pancreatin-responsive bioactive peptide, nanofiber hydrogel prepared from pancreatin-responsive bioactive peptide and application of pancreatin-responsive bioactive peptide

Abstract

The invention belongs to the technical field of cancer drugs, and particularly relates to a pancreatin-responsive bioactive peptide, nanofiber hydrogel prepared from the pancreatin-responsive bioactive peptide and application of the pancreatin-responsive bioactive peptide. The trypsin response-based bioactive peptide 1-Pept (the sequence of the trypsin response-based bioactive peptide 1-Pept is naphthylacetic acid-phenylalanine-glycine-tyrosine-lysine-cysteine-aspartic acid) and an anti-cancer drug adriamycin can be jointly assembled into 1-Pept / Dox nanofiber hydrogel, and a cytoskeleton can be destroyed by combining nanofibers with Dox; a series of cascade events resulting in cellular dysfunction are initiated, including mitochondrial damage, activation of apoptosis-associated proteins, and nuclear damage, thereby cooperatively causing tumor ablation. Compared with the existing single anti-cancer drug adriamycin, the in-vivo and in-vitro anti-cancer effect of the compound is obviously superior to that of the adriamycin pure drug, and the compound has obvious advantages in the aspect of resisting colon cancer.

Description

Technical Field

[0001] The 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 by using the same and application thereof. Background Art

[0002] Colorectal cancer is the fourth most common cancer in the world, with nearly 900,000 deaths each year. As one of the conventional treatments for colon cancer, chemotherapy has been shown to effectively alleviate the patient's condition. Doxorubicin, an antibiotic drug, can be used to treat colon cancer. However, its non-selective distribution in the body, limited response rate, and toxic side effects limit its application; more and more studies have shown that peptides with specific sequences have multiple effects: on the one hand, they can be assembled into multifunctional nanocarriers for drug delivery; on the other hand, they can play biologically active roles, including anticancer, antibacterial and anti-inflammatory effects. Enzyme-mediated peptide self-assembly (EISA) can regulate the in situ self-assembly or reassembly of peptides around cells, inside cells, and at subcellular organelle sites by overexpressing enzymes in the tumor microenvironment, thereby inducing a series of effects, such as damage to cell membrane structure, destruction of cytoskeleton structure, or dysfunction of subcellular organelles, thereby making significant progress in selectively and potently killing cancer cells. By applying the overexpressed enzyme 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 vital role in protein digestion. More and more data show that overexpressed trypsin in colon cancer is closely related to the progression of colon cancer. Therefore, the present invention uses a strategy of manipulating the in situ assembly of bioactive peptides to fight colon cancer by utilizing trypsin highly expressed in colon cancer sites. 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 by using the same and its application.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: The invention discloses a method for preparing a bioactive peptide responsive to pancreatin. The method comprises the following steps: adding dichloromethane to 2-chlorotrityl chloride resin to swell the resin, then adding condensing agents TBTU and Hobt, sequentially connecting aspartic acid, cysteine, lysine, tyrosine, glycine, phenylalanine, phenylalanine and naphthylacetic acid to the 2-chlorotrityl chloride resin until the sequence is completed, cutting the sequence from the resin with trifluoroacetic acid TFA, washing it, and freeze-drying it to obtain a crude product. The crude product is then purified with preparative HPLC to obtain a bioactive peptide 1-Pept, wherein the sequence is naphthylacetic acid-phenylalanine-phenylalanine-glycine-tyrosine-lysine-cysteine-aspartic acid, and the purity is not less than 96%.

[0006] Another object of the present invention is to provide a bioactive peptide prepared by the above preparation method.

[0007] Another object of the present invention is to utilize the prepared bioactive peptide to co-assemble with doxorubicin to prepare nanofiber hydrogel, wherein 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, wherein 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, and a morphological transformation of the disease lesion occurs.

[0008] Another object of the present invention is to provide an application of nanofiber hydrogel, which is used in the preparation of drugs for treating colorectal cancer. After the 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.

[0009] The advantages of the present invention are as follows: the 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 based on trypsin response can be co-assembled with the anticancer drug doxorubicin (abbreviated as: Dox) into 1-Pept / Dox nanofiber hydrogel (referred to as 1-Pept / Dox NFs) for effectively fighting colorectal cancer. First, a trypsin-responsive bioactive peptide 1-Pept was prepared by solid-phase synthesis technology, which includes the following parts: i) Naphthylacetic acid-phenylalanine-phenylalanine (NapFF) sequence, which can promote peptide assembly; ii) Trypsin-responsive peptide sequence Glycine-Tyrosine-Lysine-Cysteine ​​(GYKC), which can be cleaved at the carboxyl side of lysine (K); iii) C-terminal aspartic acid (D), which can bind to the anticancer drug doxorubicin (Dox) through electrostatic interaction to achieve stable loading of the anticancer drug doxorubicin. 1-Pept and Dox coassembled into injectable 1-Pept / Dox NFs through non-covalent interactions. When it came into contact with colorectal cancer sites, 1-Pept / Dox NFs showed enhanced cellular uptake through caveolae-mediated endocytosis. Subsequently, 1-Pept / Dox NFs released 1-Pept and Dox after 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 reassembled into denser Pept nanofibers (denoted as Pept NFs). Pept nanofibers combined with Dox can destroy the cytoskeleton (such as actin and tubulin), triggering a series of cascade events leading to cell dysfunction, including mitochondrial damage, activation of apoptosis-related proteins and nuclear damage, thereby synergistically causing tumor ablation. Compared with the existing single anticancer drug doxorubicin, its anticancer effect in vivo and in vitro is significantly better than that of pure doxorubicin, and has a significant advantage in fighting colon cancer.

[0010] This patent reports for the first time the bioactive peptides that respond to trypsin in colon cancer sites; the bioactive peptides with special structural design can utilize the non-covalent forces between them and chemotherapy drugs to form deformable nanostructures, which not only improves the stability of the drugs, but also enhances the intracellular uptake and anti-cancer effects; by innovating the dosage form of traditional chemotherapy drugs and developing new gel dosage forms, it is helpful to expand the scope of drug use.

[0011] Application prospects: The latest research results show that the market for gels is still in a rapid growth stage. The nanofiber gel can not only prolong the duration of drug action in vivo, but also enhance drug intracellular uptake and improve anti-cancer effects. It not only provides a new application pathway for chemotherapy drugs to fight colon cancer, but also provides a new method for developing new local drug delivery systems for disease treatment, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the TOF-MS image of 1-Pept prepared by the present invention, note, ESI-MS: C 54 H 62 N 8 O 12 S, calc.MW =1046, obsvd. [M+H] ＋ = 1047.4, [M+4H] ＋ =1050.4.

[0013] Figure 2 It is the HPLC chart of 1-Pept prepared by the present invention.

[0014] 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 N 8 O 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).

[0015] Figure 4 It is the HPLC elution curve diagram of 1-Pept, 1-Pept / Tps at 4 hours and 1-Pept / Tps at 48 hours prepared by the present invention.

[0016] Figure 5 Figure 1 is a characterization of the physicochemical properties of 1-Pept / Dox NFs. Figure A shows an optical image of 1-Pept / Dox NFs (1.0 wt% / 0.1eq) at the top. Figure A shows an optical image of "HUCM" written on 1-Pept / Dox NF using an injection needle at the bottom. Figure B shows the CAC and MGC values ​​of 1-Pept / Dox NF. Figure C shows a TEM micrograph of 1-Pept / Dox NF. Scale bar, 200 nm. Figure D shows a TEM micrograph of 1-Pept / Dox NF treated with 5.0 U / mL Tps. Scale bar, 200 nm.

[0017] Figure 6Figure 1 is a comparison of cell activity between 1-Pept / Dox NFs and free Dox solution, where Figure A is the survival rate of HT29 cells after 48 h of treatment with 1-Pept / Dox NFs or free Dox. Figure B is the IC 50 value.

[0018] Figure 7 Figure 1 is a diagram of the mechanism of action of 1-Pept / Dox NFs and free Dox solution on colon cancer cells, where Figure A is a CLSM image of the intracellular tubulin fluorescence signal after 12 h of 1-Pept / Dox NF or Dox treatment. Green is tubulin stained with TubulinTracker™ Green; blue is the cell nucleus stained with Hoechst 33342. Scale bar 20μM. Figure B is the observation of DNA damage induced by 1-Pept / Dox NFs or Dox by immunofluorescence staining. Green is Histone H2A.X foci; blue is DAPI stained nucleus. Scale bar 20μm.

[0019] Figure 8 Figure 1 is the in vitro release of 1-Pept / Dox NFs under different pH conditions, where Figure A is the cumulative release rate of Dox from 1-Pept / Dox-NF under different pH conditions, with Dox solution as a control. Figure B is the cumulative release rate of 1-Pept from 1-Pept / Dox NFs under different pH conditions.

[0020] Fig. 9 Figure 1 is a graph of the in vivo antitumor activity of 1-Pept / Dox NFs, where Figure A is the tumor growth curve of HT29 xenograft mice after treatment with PBS, 1-Pept, Dox, and 1-Pept / Dox NFs. Figure B is the H&E image of the tumors in each group. Scale bar, 100 μM. DETAILED DESCRIPTION Example

[0021] 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 times the molar amount of condensing agent TBTU and Hobt, and then adding 3 times the molar amount of Fmoc-Asp(OtBu)-OH to react for 12 hours. Then, under the action of 3 times the molar amount of condensing agent TBTU and Hobt, 6 times the molar amount of Fmoc-Cys(Mtt)-OH, Fmoc-Lys(boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, and naphthylacetic acid are added in sequence and connected to 2-chlorotrityl chloride resin in sequence until the sequence is completed, and the compound is cut 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% and its sequence was: naphthylacetic acid-phenylalanine-phenylalanine-glycine-tyrosine-lysine-cysteine-aspartic acid.

[0022] 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.

[0023] 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 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.

[0024] Another object of the present invention is to provide an application of nanofiber hydrogel, wherein the nanofiber hydrogel is used in the preparation of drugs for treating colorectal cancer to effectively fight colorectal cancer.

[0025] Experiments and applications 1.1-Synthesis and purification of Pept: 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 connected to the 2-chlorotrityl chloride resin in sequence 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 1It was characterized by H-NMR, time-of-flight mass spectrometry and reversed-phase HPLC. The purity of the obtained 1-Pept was 96%, and it could be used for subsequent experiments.

[0026] 2. Investigation of trypsin responsiveness: The structure of NapFFGYKCD (abbreviated as 1-Pept) contains the trypsin (Tps) response sequence GYKC. When Tps is overexpressed in colon cancer cells, GYKC will be converted into NapFFGYK (abbreviated as Pept). First, trypsin (1 U / mL, Tps) was co-incubated with 1-Pept solution (1 mg / mL) for enzyme catalytic kinetics experiments. At different time points, 50 uL of the solution was taken out and vortexed with 150 uL of methanol, and then filtered 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 with time. Within 72 h, more than 90% of 1-Pept was converted into Pept after Tps treatment.

[0027] 3. Preparation and Characterization of Co-assembled 1-Pept / Dox NFs 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 injectability. 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 %. In the same way, a series of 1-Pept (1.0 wt %) solutions were prepared, and 0.1 eq of Dox solution was added. It was observed within 2 hours that the MGC of 1-Pept / Dox NFs was 0.5 wt %. In addition, 1-Pept / Dox NF was prepared according to the above method, and diluted into a series of concentrations of solution. 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 (D in the middle).

[0028] 4. Cell viability assay of 1-Pept / Dox NFs HT29 cells were seeded in a 96-well plate 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 cell survival rates of the respective 1-Pept / DoxNFs and Dox pure drug solutions were calculated by measuring the absorbance. 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 noteworthy that the survival rate of HT29 cells was significantly decreased after treatment with 1-Pept / Dox NFs 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 (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.

[0029] 5. Study on the mechanism of 1-Pept / Dox NFs toxicity to colon cancer cells In order 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 h. Then the cells were treated with Tubulin Tracker™ Green (1X) working solution for 30 min. After that, the cells were stained and incubated with Hoechst 33342 (10μg / mL) for CLSM observation. Figure 7 As shown in Figure A, untreated cells exhibited well-defined microtubule structures, whereas disruption of microtubule structures was observed in cells treated with Dox and 1-Pept / Dox-NFs. In contrast, well-structured microtubule structures were barely observed in cells treated with 1-Pept / Dox NFs, which may be due to intracellular reorganization catalyzed by overexpressed Tps.

[0030] To evaluate the effect of 1-Pept / Dox NFs on the cell nucleus, HT29 cells were seeded in a laser confocal culture dish and incubated with 1-Pept / Dox (62.5μM / 6.25μM) for 12 h, and 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. After that, the cells were co-cultured 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 had almost no green fluorescence signal, while the Dox-treated cells showed bright green fluorescence intensity, indicating DNA damage. Among all groups, the cells treated with 1-Pept / Dox showed the strongest fluorescence signal intensity, which was 7.0 times and 2.3 times that of the blank control group and the Dox group, respectively, indicating that it was significantly superior to the pure Dox solution in promoting DNA damage.

[0031] 6. In vitro release behavior of Dox and 1-Pept from 1-Pept / Dox NFs 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 A, the Dox solution exhibited a burst release characteristic, and the cumulative release of Dox reached 51% within 12 h. In order to further explore the drug release mechanism, the Ritger-Peppas model was used. Calculations showed that the exponential factor n of the pure Dox solution was 0.35, indicating that the release of Dox from the free Dox solution followed the Fickian diffusion mechanism.

[0032] 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 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 the 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 the 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 in the middle). Further Ritger-Peppas model fitting showed 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, which confirmed 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 also significantly slower than that at pH 6.5 and 5.5 ( Figure 8 Figure B). At pH 7.4, 6.5, and 5.5, the calculated n values ​​of 1-Pept were 0.54, 0.55, and 0.58, respectively, which were greater than 0.43, indicating that the 1-Pept released from 1-Pept / Dox NFs exhibited non-Fickian diffusion. In conclusion, 1-Pept / Dox NFs exhibited acid-responsive properties, which may be due to the protonation of the carboxylic acid group of 1-Pept and the weakening of the electrostatic interaction between 1-Pept and Dox.

[0033] 7. Study on the anti-colorectal cancer effect of 1-Pept / Dox NFs The HT29 tumor-bearing mouse model was established to study the in vivo antitumor effect of 1-Pept / Dox NFs. HT29 cell suspension (100 μL, 5.0× 10 6The mixture was then injected subcutaneously into the right back of male BALB / C nude mice (6-8 weeks old, Jinan Pengyue Experimental Animal Breeding Co., Ltd., China). When the tumor volume approached 100 mm 3 At the time of the experiment, mice were randomly divided into four groups: (1) PBS group; (2) 1-Pept NFS; (3) free Dox solution; (4) 1-Pept / Dox NFS. 200 μL of the corresponding preparations were injected peritumorally on days 1, 4, 7, and 10. The dosages of Dox and 1-Pept were 13 μmol / kg and 130 μmol / kg, respectively. The tumor size and body weight of mice were measured every three days. According to the formula: V=L×W 2 The tumor size was obtained by 3.3 × 10 μm / 2, where L and W represent the length and width of the tumor, respectively. At the end of the 21st day, tumor tissues were collected for hematoxylin-eosin (HE) staining to observe the morphological changes of tumor cells.

[0034] 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 weak ( Fig. 9 Figure A). After calculation, the tumor volume growth rates of the PBS, 1-Pept, Dox, and 1-Pept / Dox-NFs groups on day 21 were 5.7, 4.2, 3.3, and 1.5, respectively. After treatment, H&E staining was performed on the tumor tissues. Fig. 9 As shown in Figure B, the cell density of the 1-Pept and Dox groups was slightly lower than that of the PBS group, which was consistent with the results of relative tumor volume, further demonstrating its poor tumor inhibitory effect. In particular, the tumor tissue of the 1-Pept / Dox NFs group not only showed the lowest cell density but also showed extensive necrosis, indicating that 1-Pept has a unique role in enhancing the anticancer efficacy of Dox. Therefore, 1-Pept / Dox NFs exhibited effective 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 condensation agents TBTU and Hobt are added, and 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, and the sequence is cut from the resin with trifluoroacetic acid TFA, washed, and freeze-dried to obtain a crude product, and then the crude product is 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 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 left for 2 hours to assemble into red 1-Pept / Dox NFs nanofiber hydrogel.

4. The nanofiber hydrogel prepared by the method according to claim 3 is used in the preparation of drugs for treating colorectal cancer.

Images