Peptide-based hydrogels for biological and medical applications

Abstract

The present application relates to a kind of peptide-based hydrogel with biological and medical uses.The polypeptide sequence is designed from scratch, and the peptide-based hydrogel with biological activity function is directly prepared by illuminating polypeptide aqueous solution.The polypeptide uses diphenylalanine (FF) motif as antibacterial functional unit and self-assembly driving structure, uses di-tyrosine (YY) motif as photo-crosslinking and antioxidant unit, uses epsilon-amino hexanoic acid (X) as spacer motif to regulate molecular flexibility, and balances the amphiphilicity and charge of molecule by alternating arrangement of L-aspartic acid (D) and X residue;Polypeptide precursor is subjected to photochemical reaction under the action of photosensitizer / catalyst, oxidizing agent, and three-dimensional network structure of hydrogel is formed by non-covalent interaction and covalent photo-crosslinking synergy.Graded design of alternating FF / YY structure realizes accurate regulation of gel fiberization and photochemical crosslinking degree.The obtained hydrogel not only has excellent mechanical properties, shear yielding behavior and rapid self-healing ability, but also has antibacterial, antioxidant, biocompatibility and degradability, can be firmly adhered to tissue surface, and can be widely used in biomedical fields such as wound repair, drug controlled release and tissue engineering.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a photochemically synthesized peptide-based hydrogel. This hydrogel uses diphenylalanine (FF) as the core motif for antibacterial and self-assembly, and tyrosine dimers (YY) as the photocrosslinking and antioxidant functional units. The amphiphilicity and charge balance of the molecules are regulated by the spacer motif ε-aminocaproic acid (X) and alternating L-aspartic acid (D) residues. Based on de novo design of the peptide sequence, a three-dimensional network structure is synergistically constructed using non-covalent assembly and covalent photocrosslinking, achieving precise control over the degree of gel fibrosis and photochemical crosslinking. Ultimately, a peptide-based hydrogel with precise structural controllability, flexible functional design, and good biocompatibility is obtained. Background Technology

[0002] In recent years, hydrogels have become a crucial class of soft materials, possessing extraordinary potential in various biomedical applications such as wound healing, drug delivery, and tissue engineering. Depending on their structural differences, hydrogels can be derived from covalently linked macromolecules or low-molecular-weight gels. Macromolecules typically utilize physical or chemical cross-linking units to construct robust three-dimensional networks, ensuring structural integrity and mechanical stability; while low-molecular-weight gels often employ precise molecular design to enhance fibrosis, driving innovation in biological functions. Peptides, in particular, are considered a universal platform for hydrogel design, combining the advantages of both covalent and non-covalent structures. Driven by non-covalent interactions such as hydrogen bonds, hydrophobic / hydrophilic interactions, van der Waals forces, ionic bonds, and π-π stacking, peptides can spontaneously assemble into basic structures such as ribbons, rods, sheets, and tubes. When combined with covalent strategies such as disulfide bond formation or dityrosine cross-linking, these primary peptide assemblies can transform into higher-order structures, thereby constructing hierarchically complex networks.

[0003] Based on our current research, the photochemical reaction of the YY motif exhibits extremely high cross-linking efficiency in constructing covalent networks for biofunctional peptide materials. Under visible light irradiation, linear primary peptide structures can be orderly transformed into higher-order hierarchical structures, while maintaining good functional group tolerance throughout the system, thus achieving seamless integration of different functional groups and fragments within the peptide system. De novo peptide design offers significant advantages. Its synthesis follows conventional pathways and is easy to control; the peptide sequence is encodeable, facilitating precise regulation of peptide structure and function, and possesses inherent biocompatibility, providing a powerful bottom-up strategy for material fabrication. This strategy can effectively integrate complex biological functions, enhance the bioactivity of hydrogels, and significantly improve their multifunctionality, enabling hydrogels to demonstrate superior adaptability to diverse needs in advanced biomedical applications, making them highly promising candidate materials.

[0004] Fiber fumarates (FFs), as the simplest natural fibrous framework, also possess significant antibacterial activity. This property stems from the reverse structure of FF's self-assembly motif, which effectively promotes β-sheet extension, enhances hydrophobic interactions, and strengthens aromatic π-π stacking effects. These effects collectively increase bacterial membrane permeability and promote the formation of fibrous entanglements, thereby inhibiting bacterial growth. Based on this, FFs offer a highly attractive approach for developing alternative antibacterial therapies and hold promise for opening new avenues for solving challenges in the field of antibacterial therapy.

[0005] Therefore, it is essential to design hydrogels that combine non-covalent assembly and covalent cross-linking. This approach integrates the advantages of both, enabling more complex biological functions, enhancing multifunctionality and bioactivity, and meeting diverse needs in biomedical fields such as wound repair, controlled drug release, and tissue engineering. Summary of the Invention

[0006] This invention proposes a method for preparing and applying peptide-based hydrogels by combining non-covalent assembly and covalent photocrosslinking. The core of this method lies in the de novo design of a series of amphiphilic peptides and the rapid crosslinking achieved through photochemical means to form hydrogels with excellent properties. Furthermore, this hydrogel shows broad application potential in biomedical fields such as antibacterial therapy, wound healing, controlled drug release, tissue engineering, and biosensing.

[0007] The method for preparing peptide-based hydrogels according to the present invention is characterized in that a photosensitizer and an oxidant are added during the synthesis of the hydrogel to promote the photochemical synthesis of the hydrogel. The photosensitizers used are tris(2,2'-bipyridine)ruthenium(II) chloride hexahydrate Ru(bpy)3Cl2·6H2O and phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide C. 26 H 27 O3P or 2-hydroxy-2-methyl-1-phenylpropanone C 10 H 12 O2, with ammonium persulfate (NH4)2S2O8, potassium persulfate K2S2O8, or sodium persulfate Na2S2O8 as the oxidant, and the reaction is completed within 5 minutes.

[0008] The peptide-based hydrogel of the present invention is characterized by a de novo photocrosslinkable precursor, namely a gradient design containing alternating FF / YY structures, to achieve precise control over the degree of gel fibrosis and photochemical crosslinking; and by balancing the amphiphilicity and charge distribution of the molecule through alternating arrangement of hydrophilic D and hydrophobic X residues.

[0009] The peptide-based hydrogel of this invention is characterized by using a hydrophobic FF motif as an antibacterial functional unit and a self-assembly-driven structure. Furthermore, a customized photocrosslinking YY motif provides photocrosslinking stability and antioxidant capacity.

[0010] The peptide-based hydrogel of the present invention is characterized in that the optimized polypeptide hydrogel, 4Y6F, exhibits the optimal minimum gel concentration (0.10 wt%) and mechanical properties (storage modulus of approximately 3000 Pa).

[0011] The peptide-based hydrogel of the present invention is characterized by being able to encapsulate functional molecules, wherein the encapsulated functional molecules are selected from near-infrared dyes such as Rhodamine B, Rhodamine 800, methylene blue, etc., or anticancer drugs such as paclitaxel, doxorubicin, camptothecin, etc., any one or more.

[0012] The peptide-based hydrogel of the present invention is characterized by its mechanical stability and slow degradation properties, which support its further use in articular cartilage repair or as a tissue engineering application as a hydrogel scaffold.

[0013] The peptide-based hydrogel of the present invention is characterized in that the optimized peptide-based hydrogel, especially the 4Y6F hydrogel, has antibacterial, antioxidant, and accelerated re-epithelialization processes, as well as promoting collagen deposition and angiogenesis, and its wound healing effect is significantly better than that of the control group and commercially available formulations in in vivo wound healing applications. The present invention has the following beneficial effects

[0014] Compared to non-covalently driven synthesis methods, the peptides are designed based on the principles of non-covalent assembly and covalent cross-linking, forming a stable three-dimensional network structure through the synergistic effect of self-assembly and photocross-linking. The peptides employ a gradient design with alternating FF / YY structures, allowing for precise control of gel fibrosis and photochemical cross-linking by adjusting the FF / YY ratio. Furthermore, the de novo designed peptides are prepared using a convenient standard solid-phase synthesis method, and the peptide-based hydrogel formation process is simple and easy to operate.

[0015] Compared to traditional self-assembled peptide-based hydrogels, this sequence can rapidly complete the photocrosslinking process within 5 minutes under visible light irradiation, forming a structurally stable hydrogel. Furthermore, the crosslinked network obtained by this method exhibits better stability and structural strength.

[0016] The peptide-based hydrogel of the present invention exhibits a wide range of properties, including gel self-healing, shear yielding behavior, and adhesion.

[0017] In the peptide-based hydrogel of the present invention, 4Y6F exhibits the best antibacterial effect, showing significant inhibitory effects against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Escherichia coli, demonstrating broad application potential in antibacterial therapy.

[0018] The peptide-based hydrogel of the present invention has antibacterial, antioxidant, accelerated re-epithelialization, collagen deposition and angiogenesis functions. It can inhibit the growth of pathogenic microorganisms, eliminate reactive oxygen species, enhance tissue repair, and is suitable for wound repair and promote wound healing.

[0019] The peptide-based hydrogel of the present invention has biocompatibility, biodegradability, sustained release regulation, cell adhesion and tissue repair functions, giving it potential for application in drug controlled release, tissue engineering, biosensing, flexible bioelectronic devices and regenerative medicine. Detailed Implementation

[0020] To further illustrate the present invention, a series of embodiments are given below. These embodiments are purely illustrative and are only used to specifically describe the present invention, and should not be construed as limiting the present invention.

[0021] The present invention first designed six polypeptide sequences comprising three parts: a self-assembled and antibacterial motif FF, a photocrosslinking and antioxidant unit YY, and alternating fragments of hydrophilic D and hydrophobic X residues, forming polypeptide sequences 10Y to 10F. The structure of these sequences is shown in Example 1. Attached Figure Description

[0022] Figure 1 This is a 10Y structural diagram of Example 1.

[0023] Figure 2 This is the 10Y NMR hydrogen spectrum of Example 1.

[0024] Figure 3 This is a structural diagram of the 8Y2F structure in Example 1.

[0025] Figure 4 The image shows the 8Y2F proton NMR spectrum of Example 1.

[0026] Figure 5 This is a structural diagram of the 6Y4F structure in Example 1.

[0027] Figure 6 The 6Y4F NMR hydrogen spectrum is shown in Example 1.

[0028] Figure 7 This is a structural diagram of the 4Y6F structure in Example 1.

[0029] Figure 8 The image shows the 4Y6F proton NMR spectrum of Example 1.

[0030] Figure 9 This is a structural diagram of the 2Y8F structure in Example 1.

[0031] Figure 10 The image shows the 2Y8F proton NMR spectrum of Example 1.

[0032] Figure 11 This is a 10F structural diagram of Example 1.

[0033] Figure 12 The 10F NMR hydrogen spectrum is shown in Example 1.

[0034] Figure 13 This is a transmission electron microscope (TEM) image of Example 2.

[0035] Figure 14 This is a scanning electron microscope (SEM) image of Example 2.

[0036] Figure 15 The Fourier transform infrared (FT-IR) spectrum of Example 3 is shown.

[0037] Figure 16 The image shows the fluorescence emission spectrum (FL) of Example 3.

[0038] Figure 17 This is the circular dichroism (CD) chromatogram of Example 3.

[0039] Figure 18 This is an in vitro degradation diagram of Example 4.

[0040] Figure 19 This is a rheological test diagram of Example 5.

[0041] Figure 20 The diagram shows the self-healing properties of the gel and its adhesion to tissues in Example 6.

[0042] Figure 21 This is a line graph showing the antibacterial effect of Example 7.

[0043] Figure 22 This is a diagram of in vitro free radical scavenging in Example 8.

[0044] Figure 23 This is a cell migration diagram from Example 9.

[0045] Figure 24 This is an in vivo degradation diagram of Example 10.

[0046] Figure 25 This is a diagram of wound healing in mice in Example 11.

[0047] Example 1

[0048] The designed 10Y-10F polypeptide monomers were used to manually synthesize the target polypeptide using standard solid-phase synthesis techniques. Specifically, an appropriate amount of 2-chlorotriphenylmethyl chloride resin was weighed into a polypeptide synthesis tube, and 10 mL of DMF was added to allow it to swell fully. Then, side-chain protected amino acids were sequentially loaded from the C-terminus to the N-terminus. After obtaining the desired polypeptide sequence, the sequence was first cleaved using a cleavage buffer (acetic acid:trifluoroethanol:dichloromethane = 1:2:7 v / v), followed by recrystallization and washing with icy diethyl ether to initially obtain the unprotected polypeptide. Finally, 10 mL of the deprotecting agent trifluoroacetic acid:water:triisopropylsilane = 95%:2.5%:2.5% (v / v) was added to the polypeptide powder and vortexed. After reacting for 30 minutes, the mixture was recrystallized with ice-cold ether, ground, washed, and filtered to obtain a milky white powder. The powder was labeled, weighed, and stored at 4°C. The number and position of the amino acid synthons were adjusted, and finally, six polypeptide monomers containing different phenylalanine and tyrosine, composed of alternating aspartic acid and 6-aminohexanoic acid, were synthesized.

[0049] Following the steps above, after resin activation, weigh the first amino acid at the C-terminus of the peptide (3 eq), and dissolve the catalysts 1-hydroxybenzotriazole (3 eq) and O-benzotriazole-N,N,N',N'-tetramethylhexafluorophosphate (3 eq) in 10 mL of DMF. Then add 1.1 mL of N,N-diisopropylethylamine and transfer to a peptide synthesis tube. Place the tube on a shaker and react for 3 hours. Deprotect with piperidine for 15 minutes (repeat once), wash 5 times with DMF, and then gradually introduce 3 mmol of side-chain protected tyrosine, 6-aminohexanoic acid, and aspartic acid. Repeat the coupling process to obtain the peptide reaction solution. First, cut the sequence with a cutting buffer, then recrystallize and wash with ice-cold ether to obtain the unprotected peptide. Finally, 10 mL of deprotecting agent was added to the polypeptide powder and vortexed. After reacting for 30 minutes, it was recrystallized with ice-cold ether, ground, washed, and filtered to obtain a milky white powder. Figure 1 After labeling and weighing, the sample was stored and weighed at 4°C, yielding approximately 1623 mg, with a yield of approximately 62%. Mass spectrometry and proton magnetic resonance spectrometry were measured, yielding the following data (…). Figure 2 ). HR-ESIMS:m / z=Calcd.1301.0436,found 1301.0542for[M–2H] 2– C 132 H 156 N 18 O 38 2– ;Calcd.1312.0346,found 1312.0447for[M–3H+Na] 2– . 1H NMR(600MHz,DMSO-d6,ppm)δ:12.34(br,5H),9.14(br,10H),8.14(m,2H),8.06(m,5H),7.97(m,1H),7.88(m,4H),7.69(m,4H) ,7.40(m,2H),7.01(m,20H),6.62(m,20H),4.33(m,14H),2.93(m,34H),2.38(m,2H),1.99(m,8H),1.74(s,3H),1.31(m,24H).

[0050] The synthesis method of Ac-YYXDYYDXYFFDYYDXYY-COOH(8Y2F) is similar to that of 10Y. After resin activation, the first amino acid at the C-terminus of the peptide (3 eq) and catalysts such as 1-hydroxybenzotriazole (3 eq) and O-benzotriazole-N,N,N',N'-tetramethylhexafluorophosphate (3 eq) are weighed and dissolved in 10 mL of DMF. Then, 1.1 mL of N,N-diisopropylethylamine is added dropwise, and the mixture is transferred to a peptide synthesis tube and placed on a shaker for 3 hours. Deprotection is performed with piperidine for 15 minutes (repeated once), followed by washing with DMF 5 times. Then, 3 mmol of side-chain protected tyrosine, 6-aminohexanoic acid, aspartic acid, and phenylalanine are gradually introduced, and the coupling is repeated to obtain the peptide reaction solution. The sequence is first cleaved with cleavage buffer, and then recrystallized and washed with ice-cold diethyl ether to obtain the unprotected peptide. Finally, 10 mL of deprotecting agent was added to the polypeptide powder and vortexed. After reacting for 30 minutes, it was recrystallized with ice-cold ether, ground, washed, and filtered to obtain a milky white powder. Figure 3 After labeling and weighing, the sample was stored in a 4°C refrigerator and weighed again. Approximately 1784 mg was obtained, with a yield of about 70%. Mass spectrometry and proton magnetic resonance spectrometry were measured, yielding the following data (…). Figure 4 ). HR-ESIMS:m / z=Calcd.1285.0487,found 1285.0593for[M–2H] 2– C 132 H 156 N 18 O 36 2– ;Calcd.1296.0397,found1296.0494for[M–3H+Na] 2– . 1H NMR(600MHz,DMSO-d6,ppm)δ:12.34(br,5H),9.14(br,8H),8.14(m,2H),8.02(m,6H),7.97(m,1H),7.84(m,4H),7.69(m,3H),7.39( m,2H),7.19(m,10H),7.01(m,16H),6.63(m,16H),4.33(m,14H),2.92(m,34H),2.38(m,2H),1.98(m,8H),1.74(s,3H),1.31(m,24H).

[0051] The synthesis method of Ac-YYXDFFDXYYXDFFDXYY-COOH(6Y4F) is similar to that of 10Y. After resin activation, the first amino acid at the C-terminus of the peptide (3 eq) and catalysts such as 1-hydroxybenzotriazole (3 eq) and O-benzotriazole-N,N,N',N'-tetramethylhexafluorophosphate (3 eq) are weighed and dissolved in 10 mL of DMF. Then, 1.1 mL of N,N-diisopropylethylamine is added dropwise, and the mixture is transferred to a peptide synthesis tube and placed on a shaker for 3 hours. Deprotection is performed with piperidine for 15 minutes (repeated once), followed by washing with DMF 5 times. Then, 3 mmol of side-chain protected tyrosine, 6-aminohexanoic acid, aspartic acid, and phenylalanine are gradually introduced, and the coupling is repeated to obtain the peptide reaction solution. The sequence is first cleaved with cleavage buffer, and then recrystallized and washed with ice-cold diethyl ether to obtain the unprotected peptide. Finally, 10 mL of deprotecting agent was added to the polypeptide powder and vortexed. After reacting for 30 minutes, it was recrystallized with ice-cold ether, ground, washed, and filtered to obtain a milky white powder. Figure 5 After labeling and weighing, the sample was stored in a 4°C refrigerator and weighed again. Approximately 1411 mg was obtained, with a yield of about 56%. Mass spectrometry and proton magnetic resonance spectrometry were measured, yielding the following data (…). Figure 6 ). HR-ESI MS:m / z=Calcd.1269.0538,found 1269.0526for[M–2H] 2– C 132 H 156 N 18 O 34 2– . 1H NMR(600MHz,DMSO-d6,ppm)δ:12.37(br,5H),9.14(br,6H),8.04(m,8H),7.86(m,4H),7.72(m,4H),7.52(m,2H),7.24( m,20H),6.97(m,12H),6.62(m,12H),4.51(m,14H),2.94(m,34H),2.38(m,2H),1.99(m,8H),1.74(s,3H),1.31(m,24H).

[0052] The synthesis method of Ac-FFXDYYDXFFXDYYDXFF-COOH(4Y6F) is similar to that of 10Y. After resin activation, the first amino acid at the C-terminus of the peptide (3 eq) and catalysts such as 1-hydroxybenzotriazole (3 eq) and O-benzotriazole-N,N,N',N'-tetramethylhexafluorophosphate (3 eq) are weighed and dissolved in 10 mL of DMF. Then, 1.1 mL of N,N-diisopropylethylamine is added dropwise, and the mixture is transferred to a peptide synthesis tube and placed on a shaker for 3 hours. Deprotection is performed with piperidine for 15 minutes (repeated once), followed by washing with DMF 5 times. Then, 3 mmol of side-chain protected phenylalanine, 6-aminohexanoic acid, aspartic acid, and tyrosine are gradually introduced, and the coupling is repeated to obtain the peptide reaction solution. The sequence is first cleaved with cleavage buffer, and then recrystallized and washed with ice-cold diethyl ether to obtain the unprotected peptide. Finally, 10 mL of deprotecting agent was added to the polypeptide powder and vortexed. After reacting for 30 minutes, it was recrystallized with ice-cold ether, ground, washed, and filtered to obtain a milky white powder. Figure 7 After labeling and weighing, the sample was stored in a 4°C refrigerator and weighed again. Approximately 1556 mg was obtained, with a yield of about 61%. Mass spectrometry and proton magnetic resonance spectrometry were measured, yielding the following data (…). Figure 8 ). HR-ESIMS:m / z=Calcd.1253.0589,found 1253.0655for[M–2H] 2– C 132 H 156 N 18 O 32 2– ;Calcd.1264.0499,found1264.0560for[M–3H+Na] 2– . 1H NMR (600MHz, DMSO-d6, ppm) δ: 12.34 (br, 5H), 9.15 (br, 4H), 8.20 (d, J = 7.8Hz, 2H), 8.13 (d, J = 7.8Hz, 2H), 8.04 (dt, J = 13.8and 6.0Hz,2H),7.91(d,J=8.4Hz,2H),7.81(t,J=5.1Hz,1H),7.77(t,J=5.4Hz,1H ),7.68(d,J=6.0Hz,2H),7.38(q,J=4.2Hz,2H),7.22(m,30H),7.01(d,J=8.4Hz ,4H),6.89(d,J=8.4Hz,4H),6.63(d,J=8.4Hz,4H),6.58(d,J=8.4Hz,4H),4.4 4(m,14H),2.95(m,34H),2.39(m,2H),1.96(m,8H),1.73(s,3H),1.31(m,24H).

[0053] The synthesis method of Ac-FFXDFFDXYYXDFFDXFF-COOH(2Y8F) is similar to that of 10Y. After resin activation, the first amino acid at the C-terminus of the peptide (3 eq) and catalysts such as 1-hydroxybenzotriazole (3 eq) and O-benzotriazole-N,N,N',N'-tetramethylhexafluorophosphate (3 eq) are weighed and dissolved in 10 mL of DMF. Then, 1.1 mL of N,N-diisopropylethylamine is added dropwise, and the mixture is transferred to a peptide synthesis tube and placed on a shaker for 3 hours. Deprotection is performed with piperidine for 15 minutes (repeated once), followed by washing with DMF 5 times. Then, 3 mmol of side-chain protected phenylalanine, 6-aminohexanoic acid, aspartic acid, and tyrosine are gradually introduced, and the coupling is repeated to obtain the peptide reaction solution. The sequence is first cleaved with cleavage buffer, and then recrystallized and washed with ice-cold diethyl ether to obtain the unprotected peptide. Finally, 10 mL of deprotecting agent was added to the polypeptide powder and vortexed. After reacting for 30 minutes, it was recrystallized with ice-cold ether, ground, washed, and filtered to obtain a milky white powder. Figure 9 After labeling and weighing, the sample was stored in a 4°C refrigerator and weighed again. Approximately 1556 mg was obtained, with a yield of about 63%. Mass spectrometry and proton magnetic resonance spectrometry were measured, yielding the following data (…). Figure 10 ). HR-ESIMS:m / z=Calcd.1237.0640,found 1237.0701for[M–2H] 2– C 132 H 156 N 18 O 30 2–;Calcd.1248.0549,found1248.0604for[M–3H+Na] 2– . 1 H NMR (600MHz, DMSO-d6, ppm) δ: 12.37 (br, 5H), 9.14 (br, 2H), 8.21 (d, J = 7.8Hz, 3H), 8.14 (t, J = 6.3Hz, 2H), 8.04 (m,4H),7.91(d,J=8.4Hz,1H),7.86(t,J=8.4Hz,2H),7.77(t,J=5.4Hz,1H),7.72(m,3H),7.52(dt,J=11.7and 5.7Hz,2H),7.22(m,40H),6.96(d,J=8.4Hz,4H),6.61(t,J=8.4Hz,4H),4.50 (m,14H),2.94(m,34H),2.38(m,2H),2.00(m,8H),1.73(s,3H),1.30(m,24H).

[0054] The synthesis method of Ac-FFXDFFDXFFXDFFDXFF-COOH (10F) is similar to that of 10Y. After resin activation, the first amino acid at the C-terminus of the peptide (3 eq) and catalysts such as 1-hydroxybenzotriazole (3 eq) and O-benzotriazole-N,N,N',N'-tetramethylhexafluorophosphate (3 eq) are weighed and dissolved in 10 mL of DMF. Then, 1.1 mL of N,N-diisopropylethylamine is added dropwise, and the mixture is transferred to a peptide synthesis tube and placed on a shaker for 3 hours. Deprotection is performed with piperidine for 15 minutes (repeated once), followed by washing with DMF 5 times. Then, 3 mmol of side-chain protected phenylalanine, 6-aminohexanoic acid, and aspartic acid are gradually introduced, and the coupling is repeated to obtain the peptide reaction solution. The sequence is first cleaved with cleavage buffer, and then recrystallized and washed with ice-cold diethyl ether to obtain the unprotected peptide. Finally, 10 mL of deprotecting agent was added to the polypeptide powder and vortexed. After reacting for 30 minutes, it was recrystallized with ice-cold ether, ground, washed, and filtered to obtain a milky white powder. Figure 11 After labeling and weighing, the sample was stored in a 4°C refrigerator and weighed again. Approximately 1556 mg was obtained, with a yield of about 63.6%. Mass spectrometry and proton magnetic resonance spectrometry were measured, yielding the following data (…). Figure 12 ). HR-ESIMS:m / z=Calcd.1221.0690,found 1221.0735for[M–2H] 2– C 132 H 156 N 18 O 28 2–;Calcd.1232.0600,found1232.0641for[M–3H+Na] 2– . 1 H NMR(600MHz,DMSO-d6,ppm)δ:12.37(br,5H),8.20(d,J=7.2Hz,3H),8.14(d,J =6.0Hz,2H),8.04(m,5H),7.91(d,J=8.4Hz,2H),7.81(t,J=8.4Hz,1H),7.77(t ,J=5.4Hz,1H),7.73(d,J=6.0Hz,2H),7.51(q,J=3.6Hz,2H),7.23(m,50H),4.5 0(m,14H),2.92(m,34H),2.39(m,2H),1.96(m,8H),1.73(s,3H),1.29(m,24H).

[0055] High-resolution mass spectrometry and proton nuclear magnetic resonance spectroscopy analysis showed that the actual measured values ​​of the five polypeptides were in agreement with the theoretical values, indicating that they were successfully synthesized.

[0056] Example 2

[0057] Preparation and morphology characterization of peptide-based hydrogels

[0058] Preferably, the 4Y6F sequence is used as an example. A method for preparing a stable photochemically synthesized peptide-based hydrogel includes the following steps: weighing 10 mg of peptide powder and dissolving it in 0.05 mol / L... -1 The solution was prepared in a sodium hydroxide solution, and the pH was adjusted to 5–6 to form a precursor solution. Then, freshly prepared 0.1 mol% Ru(bpy)3Cl2·6H2O solution and two equivalents of (NH4)2S2O8 solution were added, followed by gentle vortexing to ensure homogeneity. The resulting mixture was transferred to a vial and placed in a custom-made photoreactor equipped with a light-emitting diode. Under controlled intensity and current, it was irradiated with 405 nm visible light to induce photocrosslinking and form a peptide-based hydrogel.

[0059] A certain volume of the 0.5wt% peptide-based hydrogel was taken, diluted five times with deionized water, thoroughly mixed by blowing, and 50μL was dropped onto a 200-mesh carbon support film. After vacuum drying for 6 hours, TEM testing was performed. Figure 13 TEM characterization results show that the microstructure of the hydrogel is a coarse, entangled, dense nanofiber network with numerous nodes.

[0060] Preferably, the 4Y6F sequence is used as an example. A peptide-based hydrogel with a mass concentration of 2 wt% before and after photocrosslinking was prepared according to the above method. After a stable hydrogel was formed, it was frozen at -80°C for 4 hours and then freeze-dried. The freeze-dried sample was sectioned, prepared, and sputter-coated with gold for observation on SEM. Figure 14 Scanning electron microscopy results showed that the lyophilized hydrogels all exhibited a three-dimensional porous network, but after photocrosslinking, the pores were smaller (approximately 45 μm), with higher pore density and more uniform distribution.

[0061] Example 3

[0062] Characterization of the secondary structure of peptide-based hydrogels

[0063] Preferably, the 4Y6F sequence is used as an example. According to the preparation method of the hydrogel in Example 2, 1 wt% peptide-based hydrogel is prepared. After the hydrogel is stabilized, it is placed in a freezer at -80°C for 4 hours to freeze, and then placed in a freeze dryer for 3 days to freeze dry, so as to obtain the freeze-dried powder of the hydrogel. Finally, FT-IR test is performed. Figure 15 The results showed a series of significant functional group features, including hydroxyl groups (located at 3280 cm⁻¹). -1 ), alkylmethylene (located at 2930cm) -1 ), Amide I band (located at 1640 cm⁻¹) -1 ), Amide II band (located at 1515cm) -1 ), CO stretching vibration of phenyl (located at 1170 cm⁻¹) -1 ) and the bending vibration of aromatic rings (located at 800–860 cm). -1 Within the range). Further analysis of the amide I region (1600–1700 cm). -1 After performing deconvolution analysis, we found absorption peaks related to β-sheet and β-turn conformations, with the highest β-secondary structure content (34%) in 4Y6F.

[0064] Preferably, the 4Y6F sequence is used as an example. Following the hydrogel preparation method in Example 2, a 0.02 wt% peptide-based hydrogel precursor solution was prepared. Then, 0.5 mL of the gel was mixed with thiamine T solution (40 mM) in 2 mL centrifuge tubes. A control solution was prepared by diluting thiamine T with deionized water. Finally, a fluorescence emission spectrum was scanned. Figure 16 The fluorescence test results showed that, compared with the control group, the fluorescence intensity of the peptide-based hydrogel at around 483 nm was greatly enhanced, which further indicated the β secondary structure of the peptide-based hydrogel.

[0065] Preferably, the 4Y6F sequence is used as an example. Following the hydrogel preparation method in Example 2, a 0.02 wt% peptide-based hydrogel precursor solution was prepared. This solution was diluted with deionized water to a concentration of 0.0063 wt% peptide-based hydrogel and transferred to a 1 mm inner diameter cuvette for circular dichroism spectroscopy. Figure 17 As shown, the polypeptide sequences from 10Y to 10F exhibit a strong negative peak at λ≈210nm and a strong positive peak at λ≈200nm, indicating the related β secondary structure.

[0066] Example 4

[0067] Drug Encapsulation and In Vitro Degradation of Peptide-Based Hydrogels

[0068] Preferably, the 4Y6F sequence is used as an example. A method for preparing a hydrogel loaded with doxorubicin, paclitaxel, and camptothecin, and a method for studying the in vitro degradation of the hydrogel in PBS at different pH values. Following the hydrogel preparation method in Example 2, a 1 wt% polypeptide hydrogel precursor solution was prepared, and 10 μL of a 5 mg / mL solution was added. -1 The above-mentioned drugs were magnetically stirred for 5 minutes to form a homogeneous solution, which was then cured by light exposure for 5 minutes to obtain drug-loaded hydrogels. The loading rates were measured using a standard curve to be 77%, 62%, and 74%, respectively. To quantitatively assess the degradation of the 4Y6F peptide hydrogel, we monitored its mass loss during incubation in phosphate-buffered saline (PBS) at different time intervals at pH 6.0 and 7.4. Incubation was performed at 37°C, with the PBS changed every 24 hours, and the hydrogel mass was recorded daily. Figure 18 As shown, the polypeptide hydrogel exhibits good degradation in vitro.

[0069] Example 5

[0070] Rheological testing of peptide-based hydrogels

[0071] Following the preparation method of the peptide-based hydrogel in Example 2, preferably using the 4Y6F sequence as an example, the rheological properties of the peptide-based hydrogel were characterized using a rheometer. Through strain scanning tests, we determined the linear viscoelastic region within the strain range of 0.01% to 100%. Then, in the range of 0.1 to 10 rad / s... –1 Within a specific frequency range, dynamic frequency scanning was performed with a strain amplitude of 1.0% to observe how the storage modulus and loss modulus of the hydrogel changed. For testing its self-healing properties, a simple cut-and-reconnect model was used, demonstrating its good self-healing ability. Figure 19 The storage modulus of the gel was shown to be approximately 3000 Pa, which was consistently greater than the loss modulus, confirming its gel-like behavior.

[0072] Example 6

[0073] Self-healing and tissue adhesion properties of peptide-based hydrogels

[0074] Preferably, the 4Y6F sequence is used as an example. Following the hydrogel preparation method in Example 2, a 1 wt% peptide-based hydrogel was prepared. The self-healing and adhesion capabilities of the peptide-based hydrogel were primarily verified through direct visual inspection. Initially, two square hydrogel blocks were fabricated using a mold, and one of them was stained with Rhodamine B to improve its visibility. Next, these blocks were cut in half, and then the two cut parts were allowed to fuse naturally without any external intervention. After 10 minutes, the hydrogel was able to recover its initial shape. Furthermore, we investigated the adhesion properties of the peptide hydrogel to various isolated biological tissues (pig skin, mouse heart and liver, etc.). Finally, we monitored its adhesion. Figure 20 Throughout the process, the peptide-based hydrogel exhibited excellent self-healing and adhesive properties.

[0075] Example 7

[0076] Evaluation of the antibacterial effect of peptide-based hydrogels

[0077] Preferably, the 4Y6F sequence is used as an example. Following the hydrogel preparation method in Example 2, 0.125–32 mg / mL of the hydrogel was prepared. –1 Peptide-based hydrogels within a concentration range. Using Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus as test strains, we evaluated the antibacterial properties of the peptide-based hydrogels. Initially, 1 mL of peptide-based hydrogels at different concentrations were mixed with 1 mL of bacterial suspension (concentration 2 × 10⁻⁶). 6 CFU mL –1 Mix and incubate at 37°C for 8 hours. Antibacterial activity was determined using the plating method. Each sample was diluted 10⁻⁶ times. 4 The peptide-based hydrogel was evenly spread on soybean agar plates and incubated for 24 hours. Colony growth was recorded by photographing. The number of colonies in the peptide-based hydrogel treatment group and the PBS control group was compared to quantify bacterial survival. All peptide-based hydrogel treatment groups showed varying degrees of antibacterial effect. Figure 21 Furthermore, hydrogels with higher FF content exhibit better antibacterial effects. In the 8Y2F to 10F peptide series, the minimum inhibitory concentration (MIC) ranges from 32 mg / mL. –1 Reduced to 4 mg / mL –1 .

[0078] Example 8

[0079] Evaluation of free radical scavenging in vitro using peptide-based hydrogels

[0080] Preferably, the 4Y6F sequence is used as an example. Following the hydrogel preparation method in Example 2, peptide hydrogels with concentrations ranging from 0.125% to 2 wt% were prepared. In the DPPH experiment, different concentrations of peptide-based hydrogel precursor solutions and control solutions were placed in 96-well plates and incubated with an equal volume of 100 μM DPPH solution for 30 minutes, with three replicates per group. The absorbance of each group was then measured at 517 nm. Simultaneously, ABTS measurements were performed, specifically by adding 2.84 mg / mL of DPPH solution to the control solution. –1 ABTS solution with 0.664 mg / mL –1 Potassium persulfate solution was mixed in a 1:1 volume ratio and allowed to stand in the dark for 15 hours. The mixture was then diluted with anhydrous ethanol to achieve an absorbance of 0.7 ± 0.02 at 734 nm. The ABTS experiment protocol is the same as the DPPH experiment. Figure 22 Its free radical scavenging ability increases with increasing concentration and tyrosine content. When the concentration of peptide-based hydrogel is 2wt%, the scavenging rate of DPPH and ABTS free radicals exceeds 67% and 87%, respectively, indicating its good antioxidant properties.

[0081] Example 9

[0082] Evaluation of the cell migration-promoting effect of peptide-based hydrogels

[0083] Preferably, the 4Y6F sequence is used as an example. Following the hydrogel preparation method in Example 2, a 0.1 wt% peptide-based hydrogel was prepared. First, 1 × 10⁻⁶ oz. was seeded into each well of a 6-well plate. 5 NIH 3T3 cells were cultured to a 90% confluence state. Then, vertical scratches were created on the cell layer using the tip of a 200 μL pipette. The cells were then placed in DMEM medium containing 0.1 wt% peptide-based hydrogel and incubated for 24 and 48 hours, respectively. After incubation, cell migration at the scratch sites was observed using an inverted optical microscope. Figure 23 The migration area of ​​the treatment group reached 25% and 68% after 24 and 48 hours, respectively.

[0084] Example 10

[0085] In vivo degradation evaluation of peptide-based hydrogels

[0086] Preferably, the 4Y6F sequence is used as an example. Following the hydrogel preparation method in Example 2, a 1 wt% peptide-based hydrogel precursor solution was prepared, and 10 μL of a 1 mg / mL solution was added. –1A Rhodamine 800 solution was magnetically stirred for 5 minutes to form a homogeneous solution, which was then cured by light exposure for 5 minutes. The hydrogel was then implanted subcutaneously and the wound was sutured. Near-infrared fluorescence images were collected weekly postoperatively to visualize and track the degradation of the hydrogel in vivo. Figure 24 The results showed that the hydrogel completely degraded within 21 days. The mechanical stability and slow degradation properties exhibited by this peptide-based hydrogel in subcutaneous implantation support its further application in articular cartilage repair or as a tissue engineering scaffold.

[0087] Example 11

[0088] Evaluation of the therapeutic effect of peptide-based hydrogels on a mouse model of full-thickness skin defects caused by bacterial infection.

[0089] Preferably, the 4Y6F sequence is used as an example. Following the hydrogel preparation method in Example 2, a 1 wt% peptide-based hydrogel was prepared. To evaluate the antibacterial effect and wound healing performance of the peptide-based hydrogel in vivo, an 8 mm full-thickness skin defect model caused by methicillin-resistant Staphylococcus aureus (MRSA) infection was established on the back of mice. Subsequently, the mice were randomly divided into five treatment groups: PBS group, Commercial dressing group, and three hydrogel formulations containing 1 wt% peptide (4Y6F, 10F, and 10Y, respectively). We monitored wound healing using digital images taken on postoperative days 0, 3, 7, and 12. Evaluation was conducted using a mouse model of full-thickness skin defects caused by MRSA infection. Figure 25 The 4Y6F hydrogel significantly accelerated wound healing (area reduction to 18.6%, compared to 62.2%, 49.8%, 32.5%, and 38.8% for the control group, 10Y, 10F, and commercial gel, respectively).

[0090] application

[0091] This invention innovatively integrates the principles of non-covalent assembly and covalent photocrosslinking, designing an amphiphilic peptide sequence de novo and efficiently preparing a biofunctional peptide-based hydrogel using photocrosslinking technology. In the design, FF serves as the self-assembly and antibacterial core, while YY provides photocrosslinking stability and antioxidant capacity. By adjusting the FF / YY ratio, the degree of gel fibrosis and photochemical crosslinking can be precisely controlled. Alternating arrangement of hydrophilic D and hydrophobic X residues balances the amphiphilicity and charge of the molecule. This peptide system can complete rapid photocrosslinking within 5 minutes under visible light, significantly improving preparation efficiency and practicality. The resulting hydrogel not only possesses excellent mechanical properties, shear yielding behavior, and rapid self-healing ability, but also exhibits antibacterial, antioxidant, biocompatibility, and degradability, demonstrating its application potential in antibacterial therapy, wound repair, controlled drug release, and tissue engineering. In summary, this invention proposes an innovative design strategy for peptide-based hydrogels, providing a new approach for the photochemical preparation of peptide-based hydrogels and opening up new avenues for biomedical applications, demonstrating both high innovation and practicality.

[0092] The above embodiments are merely illustrative examples to clearly illustrate the present invention and are not intended to limit the implementation of the invention. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A peptide-based hydrogel with biological and medical applications, characterized in that, The polypeptide contains a hydrophobic diphenylalanine (FF) motif as an antibacterial functional group and a self-assembly driving unit; a photocrosslinked dityrosine (YY) motif as a photocrosslinking and antioxidant unit; ε-aminohexanoic acid (X) as a hydrophobic spacer to regulate the spacing between the YY and FF modules; and alternating L-aspartic acid (D) and X residues to balance the amphiphilicity and charge of the molecule. The specific sequence of the polypeptide is selected from YYXDYYDXYYXDYYDXYY(10Y) and YYXDY. YDXFFXDYYDXYY(8Y2F), YYXDFFDXYYXDFFDXYY(6Y4F), FFXDYYDXFFXDYYDXFF(4Y6F), FFXDFFDXYYXDFFDXFF(2Y8F), FFXDFFDXFFXDFFDXFF(10F); the N-terminus of the polypeptide is acetylated; the hydrogel is prepared by using the polypeptide as a crosslinking precursor and applying light irradiation in an aqueous environment.

2. The peptide-based hydrogel according to claim 1, characterized in that, The peptide is designed based on the principles of non-covalent interaction and covalent cross-linking, and forms a stable three-dimensional network structure through the synergistic effect of self-assembly and photocross-linking. The peptide adopts a gradient design with alternating FF / YY structures, and the degree of gel fibrosis and photochemical cross-linking can be precisely controlled by adjusting the FF / YY ratio.

3. The peptide-based hydrogel according to claim 1, characterized in that, During the synthesis of the hydrogel, photosensitizers and oxidants are added to promote the photochemical synthesis of the hydrogel. The photosensitizers used are tris(2,2'-bipyridine)ruthenium(II) hexahydrate Ru(bpy)3Cl2·6H2O and phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide C. 26 H 27 O3P or 2-hydroxy-2-methyl-1-phenylpropanone C 10 H 12 O2, with ammonium persulfate (NH4)2S2O8, potassium persulfate K2S2O8, or sodium persulfate Na2S2O8 as the oxidant, and the reaction is completed within 5 minutes.

4. The peptide-based hydrogel according to claim 1, characterized in that, The minimum gelation concentration of the hydrogel is 0.10wt% to 0.50wt%. The hydrogel exhibits mechanical properties with a storage modulus greater than the loss modulus, exhibits shear yielding behavior under high strain, and shows rapid self-healing ability after undergoing alternating low strain and high strain cycles. The hydrogel can restore structural integrity within 5 minutes after damage and has tissue surface adhesion, forming a stable adhesion on the surface of pig skin and mouse organs.

5. The peptide-based hydrogel according to claim 1, characterized in that, Peptide sequences containing FF exhibit significant antibacterial activity against both Gram-positive and Gram-negative bacteria. The antibacterial activity increases with increasing FF content. In the peptide series from 8Y2F to 10F, the minimum inhibitory concentration (MIC) ranges from 32 mg / mL. –1 Reduced to 4 mg / mL –1 FF promotes bacterial membrane disruption, while YY provides photocrosslinking stability and antioxidant capacity. The two work synergistically to enhance antibacterial properties. Among them, 4Y6F has the best antibacterial effect and shows significant inhibitory effects on Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Escherichia coli.

6. The peptide-based hydrogel according to claim 1, characterized in that, The hydrogel has antibacterial and antioxidant properties, inhibits the growth of pathogenic microorganisms, scavenges reactive oxygen species, is suitable for wound repair, promotes wound healing, reduces the risk of infection, and enhances tissue repair capabilities.

7. The peptide-based hydrogel according to claim 1, characterized in that, The hydrogel possesses biocompatibility, biodegradability, controlled release, cell adhesion, and tissue repair functions, and can be applied in drug controlled release, tissue engineering, biosensing, flexible bioelectronic devices, and regenerative medicine.

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