Fmoc-tyrosine / berberine chiral supramolecular antibacterial hydrogel with photodynamic effect and application thereof
By co-assembling berberine with Fmoc-Y to form a chiral supramolecular hydrogel, combined with photodynamic therapy, the problems of bacterial biofilm and antibiotic resistance were solved, achieving highly efficient antibacterial activity and biocompatibility against bacteria.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- BEIJING UNIV OF CHINESE MEDICINE
- Filing Date
- 2025-04-29
- Publication Date
- 2026-06-09
Smart Images
Figure FT_1 
Figure FT_2 
Figure FT_3
Abstract
Description
Technical Field
[0001] This invention relates to the preparation, characterization, and application of a biocompatible chiral supramolecular hydrogel with photodynamic therapy (PDT) capability. Background Technology
[0002] Bacterial infections remain a significant public health problem, contributing to high morbidity and mortality rates. Biofilms easily form when large numbers of bacteria clump together. Biofilm formation is a major cause of many chronic infections, and the reduced effectiveness of antibiotics on bacteria within the biofilm also enhances antibiotic resistance. Therefore, finding novel antimicrobial agents that are both highly effective and low in toxicity has become a global priority in the fight against bacterial infections.
[0003] Chiral assembly, an important branch of supramolecular assembly, has attracted much attention in biomedical fields such as chiral drug delivery, biosensors, and tissue engineering. Supramolecular chiral assembly is a precise and complex molecular construction process in which molecular units are stacked asymmetrically to form unique chiral supramolecular structures. This phenomenon permeates all levels of biological systems. In the biomedical field, supramolecular chiral assembly demonstrates profound application potential and value. It can influence key processes in cell culture, promote the design of antibacterial surfaces, and achieve sustained release and enantiomeric differentiation during drug delivery. Its unique advantages endow it with enormous application value and broad prospects in fields such as precision medicine and efficient drug utilization. Amino acid hydrogels, as natural antibacterial agents, possess excellent biocompatibility, biodegradability, high water content, and programmable antibacterial properties. Chiral amino acids are also ideal materials for chiral supramolecular assembly; however, research on antibacterial chiral assembly designed with amino acids is currently limited.
[0004] Photodynamic therapy (PDT) is an attractive non-invasive antibacterial and anti-biofilm treatment method with high spatiotemporal precision and low tendency for antimicrobial resistance. PDT relies on the generation of reactive oxygen species (ROS) by photosensitizers (PSs) under light irradiation to kill bacteria. Organic luminescent agents with aggregation-induced emission (AIE), or AIEgens, exhibit negligible luminescence as monomolecules, but their luminescence is enhanced when aggregated. AIEgens are also considered to enhance ROS generation, making them an important and promising class of PDT photosensitizers. Berberine (BBR), a natural isoquinoline alkaloid derived from Coptis chinensis, not only exhibits broad-spectrum antibacterial activity and multidrug resistance inhibition, but its rigid planar structure and positively charged center also endow it with unique photophysical properties and self-assembly capabilities. Notably, chiral supramolecular assembly systems, due to their unique chiral microenvironment and tunable molecular stacking modes, can effectively regulate the aggregation state and photophysical properties of photosensitizers, potentially significantly improving photodynamic antibacterial performance. Therefore, this invention plans to use the active ingredient BBR from traditional Chinese medicine and amino acids to study the synergistic effect of chiral supramolecular assembly on antibacterial and antibiofilm processes. Summary of the Invention
[0005] To address the growing challenge of antibiotic resistance globally, this invention explores the synergistic effect of berberine (BBR) in supramolecular chiral assembly. Berberine, the main active ingredient in the traditional Chinese medicine (TCM) Coptis chinensis, co-assembles with Fmoc-Y to form chiral supramolecular assemblies, effectively utilizing the chirality of amino acids and the antibacterial potential of BBR.
[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:
[0007] Berberine and Fmoc-Y were used as raw materials. Fmoc-Y powder was weighed and placed in a centrifuge tube, and dissolved in PBS. The dissolved suspension was placed in an ultrasonic machine and sonicated at room temperature for 5 minutes. Then, it was placed in a water bath and heated to 85°C until the suspension separated into layers. NaOH solution was added until the solution became clear and transparent. BBR solution was added, the centrifuge tube was removed, and the solution was left to stand at room temperature for 2 hours to obtain the Fmoc-Y / BBR hydrogel.
[0008] Fmoc-Y has two configurations: Fmoc-LY and Fmoc-DY. Using Fmoc-LY yields L-type berberine chiral supramolecular hydrogels (Fmoc-LY / BBR). Using Fmoc-DY yields D-type berberine chiral supramolecular hydrogels (Fmoc-DY / BBR).
[0009] Fmoc-Y and BBR combine in an alkaline environment and form a homogeneous, transparent solution after heating at 85°C. After cooling at room temperature for a period of time, the solution transforms into a hydrogel, exhibiting a homogeneous, yellow, and transparent appearance.
[0010] This invention also experimentally demonstrates the photodynamic therapy (PDT) capabilities of Fmoc-Y / BBR chiral hydrogel and its antibacterial applications.
[0011] The results showed that under white light irradiation, chiral hydrogels can effectively generate hydroxyl radicals (·OH) and singlet oxygen (·OH). 1 O2) enhances its antibacterial effect. When exposed to white light, the chiral hydrogels exhibit significant antibacterial and biofilm-inhibiting effects against prevalent pathogens such as Escherichia coli and Staphylococcus aureus, demonstrating differences in chiral activity. The Fmoc-LY / BBR hydrogel showed stronger antibacterial activity. Furthermore, the chiral hydrogels exhibited negligible cytotoxicity and low hemolytic activity against human immortalized keratinocytes, demonstrating their good biocompatibility.
[0012] The beneficial effects of this invention are:
[0013] This invention addresses the growing global problem of antibiotic resistance by leveraging the synergistic effect of berberine (BBR) in supramolecular chiral assembly. Berberine, the main active ingredient in TCM Coptis chinensis, co-assembles with Fmoc-Y to form a chiral supramolecular assembly, effectively utilizing the chirality of amino acids and the antibacterial potential of BBR. Under white light, the chiral supramolecular assembly effectively generates type I and type II free radicals, enhancing its antibacterial activity against common bacteria such as Escherichia coli and Staphylococcus aureus. Furthermore, the chiral hydrogel exhibits good biocompatibility, low cytotoxicity, and hemolytic activity, indicating its potential application in the field of safe biomedicine. This invention promotes the deep integration of supramolecular chiral chemistry with active ingredients in traditional Chinese medicine, opening new avenues for developing intelligent antibacterial materials with chiral recognition capabilities. Attached Figure Description
[0014] Figure 1 The chiral hydrogels Fmoc-LY / BBR and Fmoc-DY / BBR in Example 2 are characterized by SEM.
[0015] Figure 2 The circular dichroisms of Fmoc-LY / BBR and Fmoc-DY / BBR chiral hydrogels in Example 3 are shown.
[0016] Figure 3 The images show the fluorescence spectra of the Fmoc-LY / BBR hydrogel at λex = 365 nm in Example 4 and the PL spectra of Fmoc-LY / BBR at different BBR concentrations, respectively.
[0017] Figure 4 These are the CPL spectra of Fmoc-LY / BBR and Fmoc-DY / BBR in Example 4.
[0018] Figure 5 This is a material performance test of the Fmoc-LY / BBR chiral hydrogel in Example 5.
[0019] Figure 6 This is a rheological test of the Fmoc-LY / BBR chiral hydrogel in Example 5.
[0020] Figure 7 These are the DMPO ESR signals of the Fmoc-LY / BBR hydrogel ESR spectrum in Example 6 and... 1 4-oxo-TEMP ESR signal of O2.
[0021] Figure 8 This refers to the photodynamic synergistic antibacterial activity against Escherichia coli and Staphylococcus aureus in Example 7.
[0022] Figure 9 This refers to the biofilm removal rate in Example 8.
[0023] Figure 10 This refers to the in vitro drug release rates of Fmoc-LY / BBR, Fmoc-DY / BBR, and BBR in Example 9.
[0024] Figure 11 This is a hemolysis test of chiral hydrogels at different concentrations in Example 10.
[0025] Figure 12 Hacat cell viability after 24 hours of treatment with chiral hydrogel in Example 11. Detailed Implementation
[0026] Example 1: Preparation of Fmoc-Y / BBR chiral supramolecular hydrogel
[0027] 14.00 mg of Fmoc-Y powder was dissolved in 0.5 mL of PBS. The dissolved suspension was sonicated at room temperature for 5 min, and then heated in an 85°C water bath until the suspension separated into layers. 30 μL of 1 mol / L NaOH solution was added until the solution became clear. Berberine was prepared to a concentration of 1 mg / mL with deionized water, and then 0.5 mL of 1 mg / mL BBR solution was added. The mixture was allowed to stand at room temperature for 2 h to obtain Fmoc-Y / BBR chiral supramolecular hydrogel. L-type berberine chiral supramolecular hydrogel Fmoc-LY / BBR was obtained by preparing Fmoc-LY. D-type berberine chiral supramolecular hydrogel Fmoc-DY / BBR was obtained by preparing Fmoc-DY using the same method.
[0028] Example 2: Microstructure of Fmoc-Y / BBR chiral hydrogel
[0029] Fmoc-LY / BBR and Fmoc-DY / BBR hydrogel samples were diluted 50-fold, deposited on silicon wafers, and air-dried. The samples were further dried using a vacuum pump, and finally coated with a gold sputtering layer. Scanning electron microscopy (SEM) images were captured using a FEI Quantum 250 SEM with an accelerating voltage of 20 kV and an emission current of 10 μA.
[0030] like Figure 1 The microstructure of the chiral hydrogels was observed using scanning electron microscopy (SEM). Both L-type and D-type Fmoc-Y can co-assemble with BBR to form hydrogels, characterized by a three-dimensional network of chiral helical fibers. Specifically, the Fmoc-LY / BBR chiral assembly forms right-handed (p-type) helical nanoribbons with a width of approximately 100 nm and a pitch of 300-500 nm. Simultaneously, the Fmoc-DY / BBR chiral assembly forms left-handed (m-type) double-helical nanowires with a spacing of approximately 100 nm and a width of approximately 50 nm.
[0031] Example 3: Circular dichroism (CD) measurement using Fmoc-Y / BBR chiral hydrogels
[0032] The CD spectra of Fmoc-LY / BBR, Fmoc-DY / BBR, and Fmoc-Race-Y / BBR hydrogels were measured using a 200-600 (Applied Photophysics) imager. For the chiral hydrogel of Fmoc-LY / BBR, we observed a significant positive cotton effect at 225 nm, a negative peak signal at 254 nm, and a crossover signal at 231 nm. Figure 2 (Red curve). Furthermore, significant positive signals were detected at 285 nm and 351 nm. In contrast, the CD signal of the Fmoc-DY / BBR hydrogel is mirrored that of the Fmoc-LY / BBR, exhibiting opposite CD signals (…). Figure 2 (Blue curve). The racemic Fmoc-Race-Y / BBR has almost no obvious CD signal.
[0033] Example 4: AIE and CPL properties of Fmoc-Y / BBR chiral hydrogels
[0034] BBR, Fmoc-Y, and Fmoc-Y / BBR hydrogels were injected into 1 cm quartz cuvettes, and fluorescence spectra were recorded using a Hitachi F-4500 fluorescence spectrophotometer at 400 V and a slit width of 5 nm. The excitation wavelength was 365 nm. The fluorescence quantum yield was determined using a FluoroMax+ (HORIBA) fluorescence spectrometer. The CPL spectra of the chiral hydrogels were measured using a Fmoc-200 spectrophotometer (JASCO) with an excitation wavelength of 365 nm and a coverage range of 400-800 nm. The acquired data were then analyzed using JASCO's SpectraManager software.
[0035] Under ultraviolet light (365 nm), the hydrogel exhibits a bright green glow, with an observed emission wavelength of 543 nm. Figure 3 A). Furthermore, at an excitation wavelength of λex = 365 nm, when the BBR concentration in these chiral components increased from 0.5 mg / mL to 1.2 mg / mL, the photoluminescence (PL) intensity continuously increased, and the emission peak gradually red-shifted. This phenomenon confirms the existence of concentration-dependent luminescence characteristics in the components. Figure 4 Chiral hydrogels exhibited obvious circularly polarized emission (CPL) signals, with positive and negative CPL signals detected at around 545 nm for Fmoc-LY / BBR and Fmoc-DY / BBR chiral hydrogels, respectively.
[0036] Example 5: Gelation properties of Fmoc-Y / BBR chiral hydrogel
[0037] Using Fmoc-LY / BBR as the research object, 14.5 mg of the lyophilized sample powder was added to 0.5 mL each of PBS and deionized water to observe whether it exhibited a sponge effect. The sample was shaken to disrupt its structure, and after a period of time, its recovery was observed. The sample was heated to 80℃ and then cooled to observe whether it exhibited thermal reversibility. The sample was then left to stand, and its stability over 60 days was observed. Rheological measurements were performed using a rotational rheometer (DHR-2 rheometer, TA, USA).
[0038] like Figure 5 As shown, the Fmoc-LY / BBR chiral hydrogel remains stable for at least 60 days at room temperature. Furthermore, it exhibits a unique sponge effect; upon the addition of PBS / deionized water, its lyophilized powder gradually reverts to its hydrogel form. The chiral hydrogel also demonstrates good shear recovery and thermal reversibility.
[0039] like Figure 6 A series of rheological tests were conducted. Time-scan experiments showed that the storage modulus (G′) of the Fmoc-LY / BBR chiral hydrogel was consistently higher than the loss modulus (G″), confirming its good elasticity over a long period of time. Figure 6 A). Dynamic frequency scanning results show that, within the frequency range of 0–100 Hz, the G′ value of the hydrogel is consistently higher than the G″ value, highlighting its superior stability. Figure 6 B). As the oscillating strain increases, the intersection of G″ and G′ shows the shear strain capacity of the hydrogel ( Figure 6 C). Temperature rheological analysis showed that when the temperature exceeded 80℃, G″ exceeded G′, indicating the thermosensitivity of the hydrogel. Figure 6 D).
[0040] Example 6: ESR spectroscopy of Fmoc-Y / BBR chiral hydrogel
[0041] Using Fmoc-LY / BBR hydrogels as the research subject, the samples were exposed to light and dark conditions, and ESR measurements were performed on a JEOL JESFA200 spectrometer. 5,5-Dimethyl-1-pyrrolidine (DMPO) and 2,2,6,6-4-methyl-4-piperazine hydrochloride (4-oxo-TEMP) were used as ·OH and ·... 1 A specific scavenger of O2. Results showed that under white light irradiation, the ESR signal of DMPO-OH in the Fmoc-LY / BBR chiral hydrogel was enhanced, indicating increased generation of hydroxyl radicals, and a distinct four-peak signal characteristic was observed. Figure 7(Red curve). Furthermore, we observed a clear, uniformly intense three-peak signal, indicating that it was formed under white light illumination. 1 O2( Figure 7 (Blue curve). Fmoc-LY / BBR chiral hydrogels can effectively generate ·OH free radicals and under white light irradiation. 1 O2 provides the foundation for PDT therapy.
[0042] Example 7: Antibacterial test of Fmoc-Y / BBR chiral hydrogel
[0043] This study evaluated the in vitro bacterial inhibitory activity of Fmoc-Y / BBR using Gram-positive *S. aureus* (ATCC 6538P) and Gram-negative *E. coli* (ATCC25922). Fmoc-LY / BBR, Fmoc-DY / BBR, Fmoc-Race-Y / BBR, and BBR were diluted to appropriate concentrations in 48-well plates to a final volume of 500 μL per well. Then, 30 μL of *S. aureus* and *E. coli* (2 × 10⁻⁶) were added to each well. 6 (CFU / mL). In the photoinduced toxicity assay, 48-well plates were exposed to white light for 1 hour, while the control group was placed in the dark. The 48-well plates were then incubated at 37°C for 12 hours. Bacterial growth was observed using enzyme-labeled antibodies at a light density of 600 nm. Diluted bacterial suspensions were inoculated onto nutrient agar using a spread plate method and then incubated at 37°C for 14 hours to verify bacterial growth.
[0044] Figure 8 The results showed that under photodynamic therapy, the Fmoc-Y / BBR chiral hydrogel possessed broad-spectrum antibacterial properties, with significantly enhanced antibacterial effects, especially against Gram-negative bacteria. Fmoc-LY / BBR exhibited superior synergistic photodynamic antibacterial performance compared to Fmoc-DY / BBR. Plate coating also confirmed our conclusions. Colony growth results under white light irradiation showed that the antibacterial effect of the Fmoc-Y / BBR chiral hydrogel group was significantly better than other groups, confirming its significant synergistic photodynamic antibacterial activity. Furthermore, the Fmoc-LY / BBR group showed a significant advantage over the Fmoc-DY / BBR group against both Gram-positive and Gram-negative bacteria.
[0045] Example 8: Anti-biofilm test of Fmoc-Y / BBR chiral hydrogel
[0046] 200 μL of E. coli and S. aureus were incubated in 96-well plates at 37 °C for 24 h. The culture medium was then removed, and 250 μg / mL of Fmoc-LY / BBR, Fmoc-DY / BBR, and an equal volume of BBR were added for co-incubation. After 24 hours of incubation, the culture medium was removed, XTT staining solution was added, and incubation continued for 2 hours. The absorbance was measured at 450 nm.
[0047] like Figure 9 Under dark conditions, Fmoc-LY / BBR chiral hydrogels showed moderate effectiveness in eliminating biofilms of both bacteria. However, the biofilm removal capacity of Fmoc-LY / BBR chiral hydrogels was significantly enhanced under white light irradiation. Furthermore, XTT staining results observed under a fluorescence inverted microscope revealed that the control group showed a deep orange color, indicating vigorous biofilm growth. For *E. coli*, the color difference between the BBR and control groups was not significant, but under dark conditions, the color of the Fmoc-LY / BBR chiral hydrogel group was slightly lighter than that of the BBR group. However, after 60 minutes of white light incubation, the color of the Fmoc-LY / BBR chiral hydrogel groups significantly lightened to a pale yellow. Additionally, for *Staphylococcus aureus*, the Fmoc-LY / BBR chiral hydrogel groups also changed from an initial pale yellow to a protein color after light incubation, confirming that after PDT enhancement, Fmoc-LY / BBR chiral hydrogels exhibited significant biofilm eradication activity under chiral hydrogel action.
[0048] Example 9: In vitro drug release test
[0049] Two mL of sample was aspirated into a dialysis bag. Deionized water and PBS solution (pH 7.4) were mixed in a 1:1 ratio to prepare an 18 mL mixture as the drug release medium. A BBR solution of the same concentration was prepared as a control group. The dialysis bag was immersed in the release medium and stirred at 37°C and 300 rpm. At preset time points (0.5, 1, 2, 4, 6, 8, 12, and 24 h), 100 μL of sample solution was aspirated from the drug release medium for measurement. The entire experiment was repeated three times.
[0050] Figure 10 BBR exhibits a relatively rapid release rate within 0.5–2 hours, reaching equilibrium at 6 hours with a cumulative release rate of 60.72%. In contrast, the release curve of the chiral hydrogel group is relatively flat in the initial 4 hours, with slower release kinetics and a higher overall drug release rate, ultimately reaching a cumulative release rate of 80.42%, demonstrating a significant sustained-release effect.
[0051] Example 10: Hemolysis Test
[0052] Water was used as a positive control, and PBS as a negative control. The sample was mixed with red blood cells to obtain a 4% red blood cell solution. After incubation at 37°C for 1 hour, centrifugation was performed, and the absorbance at 570 nm was measured. The RHR (%) for each sample was calculated using the following formula: RHR (%) = (OD0.05)0.05 样品 -OD PBS ) / (OD 水 -OD PBS The hemolysis test results showed that the hemolysis rates of both chiral hydrogels at a concentration of 0.45 mg / mL were below the internationally recognized threshold (5%), confirming their biocompatibility. Furthermore, Fmoc-LY / BBR exhibited superior biocompatibility. Figure 11 ).
[0053] Example 11: HaCat cytotoxicity assay
[0054] HaCat cells were revived and cultured. When the cells entered the logarithmic growth phase, they were placed in 96-well plates at 5000 cells per well. The 96-well plates were incubated in a constant temperature incubator for 24 h. Fmoc-LY / BBR and Fmoc-DY / BBR hydrogels were diluted with complete cell culture medium and added to the 96-well plates at final concentrations of 50, 100, 150, and 200 μg / mL, respectively, and incubated for 24 h. The absorbance of the samples was detected at 490 nm using the MTT staining method. The inhibition rate was calculated according to the following equation: Inhibition rate % = [1 - (OD 样品 -OD 空白 ) / (OD 对照 -OD 空白 []×100%. At a concentration of 200 μg / mL, the cell inhibition rate of the chiral hydrogel was less than 10%, indicating good biocompatibility. Figure 12 ).
Claims
1. A biocompatible chiral supramolecular hydrogel with photodynamic therapy (PDT) capability, characterized in that: Using Fmoc-L-tyrosine (Fmoc-LY) or Fmoc-D-tyrosine (Fmoc-DY) and berberine (BBR) as raw materials, berberine and Fmoc-tyrosine undergo chiral supramolecular assembly to form a yellow transparent hydrogel.
2. The preparation method of the chiral supramolecular hydrogel as described in claim 1 comprises the following steps: Weighing Fmoc-LY or Fmoc-DY powder and placing it in a centrifuge tube, then dissolving it in PBS. The dissolved suspension is placed in an ultrasonic machine and sonicated at room temperature for 5 minutes, then placed in a water bath and heated to 85°C until the suspension separates into layers. NaOH solution is added until the solution becomes clear and transparent. BBR solution is added, the centrifuge tube is removed, and the solution is placed at room temperature for 1 hour to obtain the L-type berberine chiral supramolecular hydrogel (Fmoc-LY / BBR). The same procedure is used to obtain the D-type berberine chiral supramolecular hydrogel (Fmoc-DY / BBR).
3. The preparation method according to claim 2, characterized in that, The amount of Fmoc-tyrosine was 14 mg, dissolved in 0.5 mL of PBS, and the concentration of NaOH solution was 1 mol / L, with an addition volume of 30 μL.
4. The preparation method according to claim 2, characterized in that, The BBR concentration is 1 mg / mL, dissolved in deionized water. Add 0.5 mL of BBR solution.
5. The application of the photodynamic therapy (PDT) capability of the chiral supramolecular hydrogel as described in claim 1 in antibacterial applications and the chiral preference exhibited by pathogenic bacteria.