A pomegranate peel exosome-like nanoparticle and application thereof in an antibacterial and repair-promoting preparation

Abstract

The present application relates to the field of biological medicine, and more particularly to a pomegranate peel exosome-like nanoparticle and its application in antibacterial and repair promoting preparation, wherein the pomegranate peel exosome-like nanoparticle (PTP-EV) is separated from pomegranate peel juice by using differential centrifugation method combined with PEG method, and it is found by testing that the pomegranate peel exosome-like nanoparticle has good inhibition ability on staphylococcus aureus, further, the pomegranate peel exosome-like nanoparticle is combined with GelMA gel to prepare a wound antibacterial repair material, the slow release effect of the pomegranate peel exosome-like nanoparticle is realized, the local treatment effect on staphylococcus aureus infection is significantly enhanced, and wound healing is accelerated, and a new treatment strategy is provided for solving the problem of drug-resistant bacteria infection wound.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a pomegranate peel exosome-like nanoparticle and its application in antibacterial and repair-promoting agents. Background Technology

[0002] Bacterial infections of traumatic wounds, burns, or wounds caused by chronic diseases are a persistent challenge in global public health, contributing to high mortality rates and a significant economic burden. During wound healing, bacterial infections often trigger chronic inflammation, hindering the transition of the wound to the proliferative and remodeling phases, thus delaying or even halting healing. Traditional antibiotic treatment has numerous limitations, including low bioavailability, increasing antimicrobial resistance, and systemic side effects—particularly against Staphylococcus aureus, the dominant pathogen in chronic wounds. S. aureus These limitations underscore the urgent need to develop novel, naturally derived antimicrobial agents for the effective management of Staphylococcus aureus-infected wounds.

[0003] Traditional Chinese medicine (CHM) is an important source of bioactive compounds for developing therapeutic drugs. Topical application of freshly ground CHM preparations has shown significant symptom relief and wound healing effects. Among these, fresh pomegranate peel (Punicagranatum L.)—a medicinal and edible herb known for its astringent and hemostatic properties—is frequently used.

[0004] Recent advances in exosome-based therapies have opened promising new avenues for cell-free therapeutic strategies. Exosomes, naturally found in plant sap, retain the versatility of their parent cells, exhibiting significantly enhanced cellular uptake, multivalency, and synergistic effects compared to single-component therapies. However, exosome-based therapies are often limited by rapid clearance from the administration site. Biomaterial scaffolds—especially hydrogels—show great potential for exosome delivery due to their ability to locally retain, control release, and mimic the extracellular matrix (ECM). Hydrogel dressings, with their three-dimensional porous network and high water content, create ideal microenvironments for tissue repair. However, conventional hydrogels often lack mechanical toughness under dynamic wound conditions, such as in joint areas or bleeding wounds. Gelatin methacrylate (GelMA), a gelatin derivative biomaterial, rapidly forms hydrogels under mild conditions, and its structure is highly similar to the ECM, supporting epithelial regeneration and demonstrating promising application potential. Summary of the Invention

[0005] To further enhance antibacterial activity and speed up wound healing during repair, the inventors, through extensive research, proposed integrating PTP-EV (pomegranate peel exosome-like nanoparticles) into a GelMA hydrogel matrix. This allows for the preparation of an antibacterial and repair-promoting agent with sustained release and enhanced therapeutic efficacy, thus completing this invention.

[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides pomegranate peel exosome-like nanoparticles, which are prepared by differential centrifugation combined with the PEG method.

[0007] In one embodiment, the preparation method of the pomegranate peel exosome-like nanoparticles is as follows: (1) Pomegranate peel is cut into small pieces and soaked in buffer solution for a period of time, then juiced, filtered to remove impurities, and the filtrate is collected and centrifuged at 4°C to obtain the supernatant; (2) The supernatant is mixed with PEG solution, vortexed vigorously and incubated overnight at 4°C, then centrifuged at high speed, the precipitate is collected, the precipitate is resuspended and filtered sequentially through a sterile 450 nm filter membrane and a 220 nm filter membrane to finally complete the purification process and obtain pomegranate peel exosome-like nanoparticles.

[0008] In one embodiment, the gradient centrifugation is performed at speeds of 4000×g, 8000×g, 12000×g, and 18000×g, with each speed setting lasting 60 minutes.

[0009] In one embodiment, the high-speed centrifugation is performed at 4°C and 18000×g for 60 minutes.

[0010] In one embodiment, the PEG is PEG6000.

[0011] In one embodiment, pomegranate peel exosome-like nanoparticles (PTP-EVs) were extracted using differential centrifugation combined with the PEG method: Pomegranate peel was first cut into small pieces and soaked in phosphate-buffered saline (PBS) at a 1:1 mass ratio for 10 minutes. The soaked pomegranate peel was then juiced to extract a pulp, which was subsequently filtered twice through a 200-mesh sieve to remove coarse impurities. The filtrate was collected and subjected to gradient centrifugation at 4°C (centrifugation speeds of 4000×g, 8000×g, 12000×g, and 18000×g, with each speed setting for 60 minutes) to separate and remove cell debris and particulate impurities. The clarified supernatant from the centrifugation was then mixed with polyethylene glycol (PEG6000) solution at a 1:4 volume ratio, vortexed vigorously, and incubated overnight at 4°C. The precipitate was then centrifuged at 4°C and 18000×g for 60 minutes and collected. The precipitate was resuspended in 1×PBS and then filtered sequentially through a sterile 450 nm filter membrane and a 220 nm filter membrane to complete the purification process.

[0012] In a second aspect, the present invention provides an application of pomegranate peel exosome-like nanoparticles, which are used to specifically inhibit the activity of Staphylococcus aureus.

[0013] In a third aspect, the present invention provides an antibacterial repair agent loaded with pomegranate peel exosome-like nanoparticles, wherein the antibacterial repair agent is a gel dressing. In a preferred embodiment, the gel uses GelMA hydrogel as a carrier to encapsulate the aforementioned pomegranate peel exosome-like nanoparticles.

[0014] In a fourth aspect, the present invention provides the application of an antibacterial repair agent loaded with pomegranate peel exosome-like nanoparticles in the inhibition of Staphylococcus aureus.

[0015] In a fifth aspect, the present invention provides the application of an antibacterial repair agent loaded with pomegranate peel exosome-like nanoparticles in promoting wound infection repair. Preferably, the infection is a Staphylococcus aureus infection.

[0016] In this invention, the pomegranate peel exosome-like nanoparticles described herein achieve anti-Staphylococcus aureus infection by disrupting the integrity of bacterial cell membranes, inhibiting bacterial biofilm formation, and inhibiting the secretion of Staphylococcus aureus exotoxins.

[0017] In this invention, the gel dressing can release pomegranate peel exosome-like nanoparticles, inhibit the colony proliferation of Staphylococcus aureus in infected wounds, and promote the regeneration of wound granulation tissue and skin. Beneficial effects

[0018] (1) The pomegranate peel exosome-like nanoparticles provided by this invention have a significant and specific inhibitory effect on Staphylococcus aureus, and can inhibit it by... saeS-saeR This two-component system inhibits the secretion of Staphylococcus aureus exotoxin. The nanoparticles are derived from natural plants, ensuring high safety. They possess targeted antibacterial capabilities without requiring complex modifications, effectively reducing antibiotic use and the resulting drug resistance problems, while minimizing disruption to the host's microecology.

[0019] (2) The GelMA hydrogel dressing loaded with pomegranate peel exosome-like nanoparticles provided by the present invention can achieve the sustained-release effect of pomegranate peel exosome-like nanoparticles, significantly enhance its local therapeutic effect on Staphylococcus aureus infection and accelerate wound healing. The GelMA hydrogel provides physical support and a suitable microenvironment for wound repair, and works synergistically with the active ingredients of pomegranate peel exosome-like nanoparticles to jointly promote efficient regeneration and repair of infected wounds, providing a novel treatment strategy for solving the problem of drug-resistant bacterial infected wounds. Attached Figure Description

[0020] Figure 1 This diagram illustrates the morphology and average particle size of pomegranate peel exosome-like nanoparticles. In the diagram, A represents the morphology of the pomegranate peel exosome-like nanoparticles; B represents the average particle size.

[0021] Figure 2 The results of the evaluation of the specific antibacterial activity of pomegranate peel exosome-like nanoparticles are as follows: A shows the inhibition zone test results of pomegranate peel exosome-like nanoparticles against Staphylococcus aureus, Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa, and Enterobacter cloacae; B shows the colony count and plate photographs of Staphylococcus aureus under different concentrations of pomegranate peel exosome-like nanoparticles.

[0022] Figure 3 The results of the study on the mechanism of action of pomegranate peel exosome-like nanoparticles in inhibiting Staphylococcus aureus are presented. A shows the internalization and absorption of pomegranate peel exosomes by Staphylococcus aureus; B shows the SEM images of Staphylococcus aureus after treatment with pomegranate peel exosome-like nanoparticles; C shows the inhibitory effect of pomegranate peel exosome-like nanoparticles on Staphylococcus aureus biofilm formation; and D shows the RT-PCR results of the inhibition of Staphylococcus aureus exotoxin secretion by pomegranate peel exosome-like nanoparticles.

[0023] Figure 4 Preparation and characterization of GelMA hydrogel loaded with pomegranate peel exosome-like nanoparticles. In this figure, A represents the in vitro cumulative release curve of pomegranate peel exosomes from the GelMA hydrogel.

[0024] Figure 5The therapeutic effect of GelMA gel loaded with pomegranate peel exosome-like nanoparticles in a mouse model of Staphylococcus aureus infection wounds is shown in Figure A, which is a statistical graph of wound area in each group of mice; and Figure B is an HE-stained section of wound tissue. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0026] Example 1: Isolation, purification and characterization of pomegranate peel exosome-like nanoparticles (PTP-EV)

[0027] Pomegranate peel exosome-like nanoparticles (PTP-EVs) were extracted using differential centrifugation combined with the PEG method: Pomegranate peel was first cut into small pieces and soaked in phosphate-buffered saline (PBS) at a 1:1 mass ratio for 10 minutes. The soaked pomegranate peel was then juiced to extract a pulp, which was subsequently filtered twice through a 200-mesh sieve to remove coarse impurities. The filtrate was collected and subjected to gradient centrifugation at 4°C (centrifugation speeds of 4000×g, 8000×g, 12000×g, and 18000×g, with each speed setting for 60 minutes) to separate and remove cell debris and particulate impurities. The clarified supernatant from the centrifugation was then mixed with polyethylene glycol (PEG6000) solution at a 1:4 volume ratio, vortexed vigorously, and incubated overnight at 4°C. The precipitate was then centrifuged at 4°C and 18000×g for 60 minutes, and collected. After resuspending the precipitate in 1×PBS, it was filtered sequentially through a sterile 450 nm filter and then through a 220 nm filter to complete the purification process. The obtained PTP-EV was characterized using transmission electron microscopy (TEM) and a Malvern particle size analyzer. Results are as follows... Figure 1 As shown, TEM results indicate that the isolated PTP-EVs have a typical exosome structure, and particle size results show that the average particle size of PTP-EVs is 220.2 nm.

[0028] Example 2: Pomegranate peel exosome-like nanoparticles specifically inhibit the proliferation of Staphylococcus aureus.

[0029] The pomegranate peel exosome-like nanoparticles prepared in Example 1 were stored at -80 ℃ for later use.

[0030] (1) Bacterial culture and preparation Staphylococcus aureus, Escherichia coli, Enterobacter cloacae, and Micrococcus luteus were streaked onto LB agar plates and incubated at 37°C for 24 hours. The isolated colonies were aseptically transferred to LB broth and incubated in a shaking incubator (220 rpm, 37°C) for subsequent experiments.

[0031] (2) Antibacterial activity test method a. Agar well diffusion method Bacterial suspension (1×10) 8 Particles (CFU / mL, 50 μL) were evenly spread onto LB agar plates. Aseptically punched wells (6 mm in diameter) and 70 μL of PTP-EV solution was added to each well. The plates were incubated at 37°C for 24 hours. The diameter of the inhibition zone (including the well) was measured using digital calipers. A negative control (wells containing PBS) was included, and the experiment was repeated three times.

[0032] b. Plate counting method PTP-EV with bacterial suspension (1×10⁻⁶) 7 The PTP-EV mixtures (CFU / mL) were thoroughly mixed to achieve final concentrations of 5.68, 2.84, and 1.42 mg / mL, respectively. An equal volume of phosphate-buffered saline (PBS) was added to the control group bacteria. All samples were incubated at 37°C for 4 hours. The mixtures from each treatment group were then inoculated onto LB agar plates and incubated at 37°C for 24 hours. The viable cell count was then performed. Each test was repeated three times.

[0033] (3) Experimental results See Figure 2 PTP-EV exhibited selective antibacterial activity against Staphylococcus aureus, forming a clear inhibition zone with a diameter of 15.23 mm, while showing no inhibitory effect on other tested bacterial species. Plate count results showed that the bactericidal effect of PTP-EV against Staphylococcus aureus was concentration-dependent: within 240 minutes, the viable count of Staphylococcus aureus decreased by 2.48 LogCFU / mL, and the antibacterial activity reached 99% compared with the control group, indicating that it has rapid and potent antibacterial activity.

[0034] Example 3: Study on the mechanism by which pomegranate peel exosome-like nanoparticles inhibit the proliferation of Staphylococcus aureus

[0035] (1) Absorption of pomegranate peel exosome-like nanoparticles by Staphylococcus aureus With a concentration of 1×10 7CFU / mL of bacteria were mixed with 1 mg of DIO-labeled pomegranate peel exosome-like nanoparticles and incubated under suitable conditions for 4 hours. After incubation, the mixture was transferred to a 100 kD ultrafiltration tube and ultrafiltered at 15,000 rpm for 10 minutes to remove DIO dye adhering to the bacterial wall; the sample was then washed twice with PBS. The bacteria were then suspended in 0.5% Triton X-100 solution for 8 minutes and washed three times with PBS. The uptake of pomegranate peel exosome-like nanoparticles by the bacteria was observed using confocal microscopy.

[0036] (2) Bacterial morphological characterization Fix the cell slide to the bottom of the 12-well plate and adjust the bacterial suspension concentration to 1×10⁻⁶. 6 CFU / mL, add 2 mL of diluted bacterial suspension to each well, and incubate at 37°C for 24 hours to promote biofilm formation. Replace with LB medium containing PBS and PTP-EV, and continue incubation at 37°C. Fix samples overnight with 2.5% glutaraldehyde at 4°C, then dehydrate stepwise with a gradient of ethanol (10%, 30%, 50%, 70%, 90%, 100%), 10 minutes per step; after drying with nitrogen, spray with gold film and perform SEM imaging.

[0037] (3) Biomembrane inhibition experiment Adjust the bacterial suspension concentration to 1×10 6 CFU / mL, after culturing for 24 hours, aliquots were transferred to 24-well plates and cultured for another 24 hours to promote biofilm formation. The medium was then replaced with fresh LB medium supplemented with PBS and PTP-EV, and cultured for another 24 hours. The biofilm was fixed with methanol for 15 minutes, air-dried at room temperature, and stained with 0.1% (w / v) crystal violet for 10 minutes. Representative images from each well were collected for qualitative analysis. The wells were washed twice with PBS to remove unbound dye. The bound crystal violet was dissolved in 33% (v / v) glacial acetic acid, and the absorbance was measured at 590 nm using a microplate reader to quantify the biofilm biomass.

[0038] (4) RT-PCR experiment A suspension of *Staphylococcus aureus* in the logarithmic growth phase was incubated with 12 mg / mL pomegranate peel exosome-like nanoparticles (PTP-EV) solution at 37°C for 4 h. A PBS treatment group without PTP-EV was used as a negative control. Each experiment was independently repeated three times. After incubation, total RNA was extracted from the bacteria in each group using the Trizol method. The 16S rRNA gene was used as an internal control. saeS and saeRThe gene was used as the target gene, and quantitative PCR reaction and instrument detection were performed using the SYBR Premix Ex Taq™ kit.

[0039] (5) Test results All data were analyzed using one-way ANOVA and Tukey post-hoc test in GraphPad Prism 8 software for multiple comparisons. p <0.05,** P <0.01, *** P A value <0.001 was considered statistically significant. Based on the Ct value of the internal reference 16S rRNA, relative quantification was performed using formula 2. -ΔΔCt Calculate and analyze the fold change in relative expression levels.

[0040] See Figure 3 Confocal microscopy revealed significant co-localization between DIO-labeled PTP-EVs and Staphylococcus aureus cells, indicating that the nanoparticles could be efficiently internalized by bacteria. Biofilm inhibition experiments showed that treatment with 5.68 mg / mL PTP-EVs reduced bacterial biofilm formation by 89.28%. Scanning electron microscopy (SEM) further revealed significant morphological changes in PTP-EV-treated Staphylococcus aureus, including cell membrane shrinkage, deformation, and rupture. Furthermore, RT-PCR analysis showed that in the experimental group… saeS and saeR The gene expression level was significantly lower than that of the control group, indicating that pomegranate peel exosome-like nanoparticles can inhibit gene expression. saeS-saeR A two-component signaling system effectively inhibits the secretion of Staphylococcus aureus exotoxin.

[0041] Example 4: Preparation of GelMA gel loaded with pomegranate peel exosomes and sustained release of exosomes

[0042] (1) Preparation of GelMA gel loaded with pomegranate peel exosomes A 10% (w / v) solution of methacrylamide gelatin, a 0.4% (w / v) solution of phenyl (2,4,6-trimethylbenzoyl) lithium phosphate, and a 12 mg / ml PTP-EV protein solution were mixed at a volume ratio of 2:1:1 and stirred thoroughly until homogeneous. The mixture was then placed 5 cm away from a 405 nm UV light source and irradiated for 15-20 seconds to obtain a PEV-GM hydrogel.

[0043] (2) Detection of sustained-release properties of exosomes Add 150 μL of PEV-GM solution containing 500 μg of exosomes to each well of a 24-well plate; then add 500 μL of phosphate-buffered saline (PBS) to each well containing the gel. Collect PBS every 12 hours, replenishing with fresh PBS immediately after each collection. Measure the protein concentration in the collected PBS at each time point using a BCA kit to quantify the amount of exosomes released.

[0044] (3) Test results See Figure 4 After being embedded in exosomes, the hydrogel maintains a uniform three-dimensional network structure with uniformly distributed and interconnected pores. The PEV-GM hydrogel has good structural integrity and can support the continuous release of exosomes, with a cumulative release rate of approximately 52.14% within 72 hours.

[0045] Example 5: The therapeutic effect of GelMA gel loaded with pomegranate peel exudate-like nanoparticles on Staphylococcus aureus-infected wounds in mice.

[0046] (1) Grouping of experimental animals and establishment of a Staphylococcus aureus infection wound model Male BALB / c mice (6 weeks old, weighing 18-22g) were selected and randomly divided into 3 groups after one week of acclimatization feeding. Untreated control group, blank gel group (GelMA), and exosome-loaded gel group (PEV-GM). After anesthesia with sodium pentobarbital, a full-thickness circular dorsal wound (8 mm in diameter) was surgically created in each mouse. Each wound was inoculated with 20 μL of Staphylococcus aureus suspension (10 μL / mL). 8 CFU / mL) and incubated for 6 hours to establish infection. Postoperatively, the GelMA gel group and the PEV-GM gel group received daily topical gel dressings (dressings changed every 4 days), while the control group received no treatment. Wound healing was monitored using digital imaging, and wound area was quantified using ImageJ software. On day 12 post-infection, wound tissue was collected, fixed with 4% paraformaldehyde, and stained with H&E.

[0047] (2) Test results See Figure 5Mice treated with PEV-GM hydrogel showed significantly smaller wound areas at all time points compared to the groups treated with GelMA hydrogel alone and the control group, with a marked reduction in yellow exudate. Skin biopsy specimens taken on day 12 post-infection, stained with hematoxylin and eosin (H&E), showed significant granulation tissue formation and skin regeneration in the PEV-GM hydrogel-treated group; in contrast, the groups treated with GelMA hydrogel alone and the control group still exhibited persistent tissue defects, with only early signs of collagen deposition observed. These histological results are consistent with macroscopic observations of wound healing.

[0048] The test results of this invention demonstrate that pomegranate peel exosome-like nanoparticles and their GelMA-loaded gel dressings exhibit clear effects in specific antibacterial activity and promoting wound healing. This technical solution, derived from natural products, possesses significant advantages such as high safety, low likelihood of inducing drug resistance, and dual antibacterial and repairing efficacy. Therefore, this invention lays a solid foundation for the development of novel topical drugs or medical dressings targeting Staphylococcus aureus infections, with broad market prospects.

[0049] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. The application of pomegranate peel exosome-like nanoparticles in the preparation of antibacterial agents, characterized in that, The antibacterial activity is to inhibit Staphylococcus aureus. The preparation method of the pomegranate peel exosome-like nanoparticles includes: (1) cutting the pomegranate peel into small pieces and soaking it in a buffer solution for a period of time, then juicing it, filtering to remove impurities, collecting the filtrate and performing gradient centrifugation at 4°C, and taking the supernatant; (2) mixing the supernatant with PEG solution, vortexing it vigorously and then incubating it overnight at 4°C, then centrifuging it at high speed, collecting the precipitate, resuspending the precipitate, and filtering it through a sterile 450 nm filter membrane and a 220 nm filter membrane in sequence to finally complete the purification process and obtain pomegranate peel exosome-like nanoparticles.

2. The application according to claim 1, characterized in that, The gradient centrifugation was performed at speeds of 4000×g, 8000×g, 12000×g, and 18000×g, with each speed setting lasting 60 minutes.

3. The application according to claim 1, characterized in that, The high-speed centrifugation was performed at 4°C and 18000×g for 60 minutes; the PEG was PEG6000.

4. The application of pomegranate peel exosome-like nanoparticles in the preparation of antibacterial and repair-promoting agents loaded with pomegranate peel exosome-like nanoparticles, characterized in that, The antibacterial and repair-promoting agent is a gel dressing, which contains pomegranate peel exosome-like nanoparticles. The antibacterial agent is Staphylococcus aureus. The preparation method of the pomegranate peel exosome-like nanoparticles includes: (1) cutting pomegranate peel into small pieces and soaking them in buffer solution for a period of time, then juicing them, filtering to remove impurities, collecting the filtrate and performing gradient centrifugation at 4°C, and taking the supernatant; (2) mixing the supernatant with PEG solution, vortexing it vigorously, and incubating it overnight at 4°C, then centrifuging it at high speed, collecting the precipitate, resuspending the precipitate, and filtering it through a sterile 450 nm filter membrane and a 220 nm filter membrane in sequence to finally complete the purification process and obtain pomegranate peel exosome-like nanoparticles.

5. The application according to claim 4, characterized in that, The gel excipient uses GelMA hydrogel as a carrier.

Images