Preparation method and application of albumin nanoparticles loaded with phenolic network
By preparing albumin nanoparticles loaded with phenolic networks, the problems of low bioavailability and poor targeting of epigallocate gallate are solved, precise delivery of neutrophils and improvement of the inflammatory microenvironment are achieved, and alveolar bone regeneration and periodontal tissue repair are promoted, providing an effective nanotherapy strategy.
Patent Information
- Application Number
- CN202510679091.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-26
- Publication Date
- 2025-08-19
AI Technical Summary
The prior art is difficult to achieve the precise delivery of epigallocate gallate, which has problems such as low bioavailability, poor targeting and inability to pass through biological barriers, and the role of neutrophils in periodontitis has not been fully utilized.
Albumin nanoparticles (BEC NPs) loaded with phenolic networks, and by loading epigallocate-copper (EGCG-Cu) metal phenolic networks (EC NPs) into bovine serum albumin nanoparticles (BSANPs), an intelligent nanodelivery system was constructed to specifically target neutrophils at the inflammatory site, release EC NPs in response to the inflammatory microenvironment, eliminate reactive oxygen species and inhibit the formation and release of extraneutrophil traps, promote macrophages to M2 type polarization, and inhibit the pyroptosis pathway.
It significantly improves the inflammatory microenvironment, promotes alveolar bone regeneration and periodontal tissue repair, has good biocompatibility and no systemic toxicity, and provides nanotherapy strategies for anti-inflammatory, immune regulation and tissue repair.
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Figure CN120501722A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a preparation method and application of albumin nanoparticles loaded with a phenolic network. Background Art
[0002] Periodontitis is a chronic inflammatory disease caused by dental plaque biofilms, resulting in extensive destruction of periodontal supporting tissues. It poses a significant economic burden worldwide. Currently, treatments for periodontitis primarily include surgical procedures such as supragingival scaling, subgingival curettage, and periodontal surgery, or medications such as antibiotics. However, these approaches are largely ineffective in improving periodontal dysfunction. In-depth investigation of its pathogenesis reveals that inflammatory senescence of the periodontium, a state of permanent cell cycle arrest, is the primary cause of periodontal dysfunction. During periodontal aging, a chronic, systemic proinflammatory state develops, with aging and inflammation interacting to form a vicious cycle of tissue damage. Studies have shown that macrophages are the primary regulators of inflammatory senescence. Macrophage pyroptosis activates the cleavage of gasdermin D, which in turn forms pores in the cell membrane. Numerous proinflammatory cytokines are released from these pores into the surrounding tissue, inducing fibroblast senescence. The decreased proliferation and viability of aging gingival fibroblasts severely impairs periodontal ligament function and exacerbates periodontitis. Neutrophils have also been shown to be key players in chronic periodontitis, with established animal model studies demonstrating that neutrophils infiltrate the oral mucosa at early stages of periodontitis induction. Furthermore, neutrophil extracellular traps (NETs) are an early trigger of pathogenic inflammation in periodontitis; their excessive release stimulates the activation of macrophage pyroptosis and mediates a severe inflammatory cascade. Therefore, inhibiting the abnormal release of NETs, and thereby inhibiting macrophage pyroptosis, is of great significance for the early improvement of inflammatory aging in periodontitis.
[0003] Epigallocatechin gallate (EGCG), an active ingredient in tea, possesses anti-inflammatory, antioxidant, and vascular protective properties. It has been shown to be an effective NETs inhibitor, inhibiting the release of neutrophil myeloperoxidase (MPO) and neutrophil elastase (NE). When injected directly into the area of periodontitis, EGCG is susceptible to oxidative inactivation, resulting in poor stability and low bioavailability. Hong et al. demonstrated that constructing epigallocatechin gallate-copper (EGCG-Cu) coordination complexes (EC NPs) not only improves the stability of EGCG but also effectively promotes its anti-inflammatory and antioxidant properties. However, due to the presence of abundant saliva in the periodontal environment and susceptibility to food friction, the nanoparticles have difficulty residing in the gingival sulcus.
[0004] Nanoparticles can be internalized into neutrophils through molecules mediated by neutrophil surface molecules and delivered to target tissues via hitchhiking on these neutrophils. Albumin nanoparticles, due to their biodegradability, nontoxicity, nonimmunogenicity, and ease of preparation and reproducibility, are ideal nanodrug carriers and have been approved for clinical use by the US Food and Drug Administration (FDA). Studies have shown that BSA nanoparticles can be delivered to sites of acute inflammation by hitchhiking on neutrophils. Bovine serum albumin nanoparticles (BSANPs) can be internalized by neutrophils by binding to Fcγ receptors highly expressed on the neutrophil membrane. Desolvation promotes the formation of Schiff base bonds between the two carbonyl termini of glutaraldehyde and the amino groups of proteins. Under inflammatory conditions, these Schiff base bonds can be cleaved in response to the acidic environment within neutrophils.
[0005] At present, although most improved epigallocatechin gallate application methods can improve its bioavailability to a certain extent, there are still problems such as low loading rate, poor targeting, and inability to pass through biological barriers. Achieving precise delivery of epigallocatechin gallate while improving bioavailability, ensuring good biocompatibility and stable therapeutic effect is of great significance to promoting its clinical application. In addition, neutrophils, as key cells for immune regulation, have been widely used in experimental research, and delivery systems using them as carriers have also attracted much attention. However, most existing studies only utilize the migration characteristics of neutrophils, but ignore their complex internal environment changes and their "double-edged sword" effects. In view of this, the present invention proposes a preparation method and application of albumin nanoparticles loaded with a phenolic network, aiming to prepare an intelligent nanosystem that can respond to programmed microenvironmental changes of neutrophils, and realize spatiotemporal regulation of the progression of periodontitis. Summary of the Invention
[0006] The purpose of the present invention is to provide a preparation method and application of albumin nanoparticles loaded with phenolic network, aiming to solve the problems raised in the above background technology.
[0007] The purpose of the present invention is achieved through the following technical solutions:
[0008] A method for preparing albumin nanoparticles loaded with a phenolic network comprises the following steps:
[0009] Step 1: Dissolve epigallocatechin gallate in anhydrous ethanol and 2+ The salt was dissolved in anhydrous ethanol, and then the Cu 2+ The ethanol solution of salt was slowly added dropwise to the ethanol solution of EGCG at a volume ratio of 3:1 and stirred to form a homogeneous solution;
[0010] Step 2: The pH of the homogeneous solution was adjusted to 7.0 by dropwise addition of sodium hydroxide solution and the mixture was reacted at 37°C for 24 hours. After the reaction, the complex in the mixture was harvested by centrifugation, washed with ethanol, and finally dried under vacuum to obtain the EGCG-Cu metal phenolic network, i.e., EC NPs.
[0011] Step 3: Completely dissolve bovine serum albumin in ultrapure water, and then completely dissolve EC NPs in the bovine serum albumin solution;
[0012] Step 4: The aqueous solution obtained in step 3 was vigorously stirred at 25°C and anhydrous ethanol solution was injected to form a colloidal solution;
[0013] Step 5: After 15 min, glutaraldehyde solution was added dropwise with continuous stirring to crosslink the molecules and form bovine serum albumin nanoparticles, i.e., BSANPs.
[0014] Step 6: After 24 h of reaction, the precipitate was collected by centrifugation, washed with PBS solution, and freeze-dried for 48 h to obtain albumin nanoparticles loaded with phenolic network, namely BEC NPs.
[0015] Furthermore, the Cu 2+ The salt is CuCl2·2H2O.
[0016] Furthermore, the injection rate of the anhydrous ethanol solution is 2 mL / min.
[0017] The invention discloses albumin nanoparticles loaded with phenolic network, which are prepared by the above-mentioned preparation method.
[0018] A use of the above-mentioned albumin nanoparticles loaded with phenolic network in the preparation of a drug for treating chronic periodontitis, wherein the BEC NPs act through the following mechanism:
[0019] Specific targeting of neutrophils at sites of inflammation;
[0020] EC NPs are released in response to the inflammatory microenvironment, scavenging reactive oxygen species and inhibiting the formation and release of neutrophil extracellular traps;
[0021] Promote macrophage polarization to M2 type, downregulate pro-inflammatory factors TNF-α, IL-1β, IL-6 and upregulate anti-inflammatory factors IL-10, Arg-1, TGF-β;
[0022] Inhibit cell pyroptosis pathway and reduce inflammatory damage;
[0023] Ultimately, it can promote alveolar bone regeneration and periodontal tissue repair.
[0024] Furthermore, the BEC NPs were administered at a concentration of 250 μg / mL by local injection.
[0025] A pharmaceutical composition comprising the above-mentioned albumin nanoparticles loaded with phenolic network and a pharmaceutically acceptable carrier, for treating chronic periodontitis.
[0026] Furthermore, the pharmaceutical composition is administered by local injection.
[0027] Compared with the prior art, the present invention has the following beneficial effects:
[0028] The present invention constructs an intelligent nanodelivery system (BEC NPs) by loading epigallocatechin gallate-copper (EGCG-Cu) metal phenolic networks (EC NPs) into bovine serum albumin nanoparticles (BSANPs). BECNPs can specifically target neutrophils at the site of inflammation and release EC NPs on demand in response to the inflammatory microenvironment. They significantly improve the inflammatory microenvironment by efficiently scavenging reactive oxygen species and inhibiting the formation and release of neutrophil extracellular traps (NETs). In addition, BECNPs effectively alleviate inflammatory damage by promoting macrophage polarization to M2 type, downregulating proinflammatory factors and upregulating anti-inflammatory factors, and inhibiting cell pyroptosis pathways. In vivo experiments further demonstrated that BEC NPs can significantly promote alveolar bone regeneration and repair periodontal tissues, and have good biocompatibility and no systemic toxicity. The present invention provides a spatiotemporally regulated nanotherapy strategy for chronic periodontitis that combines anti-inflammatory, immunomodulatory and tissue repair functions, and has important clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1 Microscopic images of EC NPs and BEC NPs loaded with EC NPs; a is the transmission electron microscopy image of EC NPs, b is the transmission electron microscopy image of BEC NPs, and c is the elemental mapping of N, S and Cu elements in BEC NPs.
[0030] Figure 2 These are the particle size analysis results of EC NPs and BEC NPs.
[0031] Figure 3 is the Zeta potential of EC NPs, BSANPs, and BEC NPs.
[0032] Figure 4 Fourier transform infrared spectra of EGCG, Cu, EC NPs, BSANPs, and BEC NPs.
[0033] Figure 5 UV-visible-near-infrared spectra of EC NPs, BSANPs, and BEC NPs.
[0034] Figure 6 The results of cck8 cell activity test with BEC NPs application concentrations of 0μg / mL, 125μg / mL, 250μg / mL, 375μg / mL, 500μg / mL, and 625μg / mL.
[0035] Figure 7 Live-dead staining results of BEC NPs at concentrations of 0 μg / mL, 125 μg / mL, 250 μg / mL, 375 μg / mL, 500 μg / mL, and 625 μg / mL (scale bar = 100 μm).
[0036] Figure 8 ROS scavenging experiments and quantitative analysis of EC NPs and BEC NPs.
[0037] Figure 9 Quantitative polymerase chain reaction (qPCR) was used to detect the messenger RNA (mRNA) expression levels of M1 phenotype-related factors: TNF-α, IL-1β and the messenger RNA (mRNA) expression level of M2 phenotype-related factor: Arg-1 in RAW 264.7 cells.
[0038] Figure 10 The percentages of CD86+ and CD206+ cells in EC NPs and BEC NPs were determined by flow cytometry; a is the percentage of CD86+ cells in different treatment groups, and b is the percentage of CD206+ cells in different treatment groups.
[0039] Figure 11 Fluorescence images showing the efficient uptake of BEC NPs by neutrophils (scale bar = 20 μm).
[0040] Figure 12 SYTOX Green staining, Annexin V-mcherry staining and quantitative analysis of neutrophils after treatment with BSANPs and BEC NPs (scale bar = 20 μm).
[0041] Figure 13 Immunofluorescence staining and quantitative analysis of neutrophil extracellular trap (NETs)-associated proteins MPO and NE after treatment with NAC (ROS inhibitor) and BEC NPs (scale bar = 20 μm); a is the immunofluorescence staining image of NETs-associated protein NE in different treatment groups, b is the immunofluorescence quantitative analysis of NETs-associated protein NE in different treatment groups, c is the immunofluorescence staining image of NETs-associated protein MPO in different treatment groups, d is the immunofluorescence quantitative analysis of NETs-associated protein MPO in different treatment groups.
[0042] Figure 14 Immunofluorescence staining results (scale bar = 50 μm) and quantitative analysis of inflammatory-related cytokines TNF-α, IL-6, IL-10, and Arg-1 in macrophages treated with BSANPs and BEC NPs.
[0043] Figure 15 Quantitative polymerase chain reaction (qPCR) was used to detect the messenger RNA (mRNA) expression levels of M1 phenotype-related factors: IL-6, TNF-α, IL-1β and the messenger RNA (mRNA) expression levels of M2 phenotype-related factors: Arg-1, IL-10, TNF-β in macrophages treated with BSANPs and BEC NPs.
[0044] Figure 16 Quantitative polymerase chain reaction (qPCR) was used to detect the expression levels of pyroptosis-related cytokine mRNA (IL-1β, IL-18, caspase1, NLRP3, GSDMD) in macrophages treated with BSANPs and BEC NPs.
[0045] Figure 17 To detect macrophage pyroptosis by measuring LDH content.
[0046] Figure 18 CT images of the maxillary second molars of mice with periodontitis in different treatment groups.
[0047] Figure 19 These are the local HE and Masson staining results of the maxillary second molars of mice with periodontitis in different treatment groups; a is the HE staining result; b is the Masson staining result.
[0048] Figure 20 HE staining results of the heart, liver, spleen, lung, and kidney of mice with periodontitis in different treatment groups (scale bar = 100 μm). DETAILED DESCRIPTION
[0049] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.
[0050] The specific implementation of the present invention is described in detail below with reference to specific embodiments.
[0051] Example 1: Preparation and characterization of albumin nanoparticles loaded with phenolic network;
[0052] A method for preparing albumin nanoparticles loaded with a phenolic network comprises the following steps:
[0053] Step 1: Dissolve 1 μmol epigallocatechin gallate (EGCG) in 1 ml of anhydrous ethanol and 6 μmol Cu 2+ The salt (CuCl2·2H2O) was dissolved in 1 ml of anhydrous ethanol, and then the Cu 2+ The ethanol solution of (CuCl2·2H2O) was slowly added dropwise to the ethanol solution of EGCG with a volume ratio of 3:1 and stirred to form a homogeneous solution;
[0054] Step 2: Carefully add sodium hydroxide solution dropwise to adjust the pH of the homogeneous solution to 7.0, and react at 37°C for 24 hours. After the reaction, centrifuge at 6000 rpm for 5 minutes to harvest the complex in the mixture, wash it twice with PBS, and freeze it in a -80°C refrigerator overnight. Then, transfer it to a freeze dryer and freeze-dry it for 48 hours to obtain EC NPs (EGCG-Cu coordination compound, i.e., EGCG-Cu metal phenolic network).
[0055] Step 3: Dissolve 25 mg of BSA (bovine serum albumin) completely in 1 mL of ultrapure water, and then dissolve 4 mg of EC NPs completely in the BSA solution.
[0056] Step 4: The aqueous solution obtained in step 3 was vigorously stirred at 25°C, and 4 ml of anhydrous ethanol solution was injected at a rate of 2 mL / min to promote the solution to form a colloidal solution;
[0057] Step 5: After 15 min, add 100 μL of 8% glutaraldehyde solution dropwise with continuous stirring to crosslink the molecules to form BSANPs (bovine serum albumin nanoparticles);
[0058] Step 6: After 24 hours of reaction, centrifuge at 12,000 rpm for 20 minutes at 4°C, collect the precipitate, wash it three times with PBS solution, and freeze it in a -80°C refrigerator overnight. Then transfer it to a freeze dryer and freeze-dry it for 48 hours to obtain albumin nanoparticles loaded with phenolic network, namely BEC NPs.
[0059] like Figure 1 As shown in Figures ac, EC NPs are irregular spherical, BEC NPs are regular spherical, and irregular spherical EC NPs can be observed inside the BEC NPs. The successful synthesis of BEC NPs is proved by the element mapping of N, S, and Cu.
[0060] The particle size analysis results of EC NPs and BEC NPs are shown in Figure 2. Figure 2 As shown, the results showed that the particle size of EC NPs was about 370 nm and the particle size of BEC NPs was about 456.9 nm.
[0061] The zeta potential of EC NPs, BSANPs and BEC NPs is shown in Figure 2. Figure 3 As shown, the results showed that both EC NPs and BSA NPs were negatively charged, and as EC NPs were loaded onto BSANPs to form BEC NPs, the negative charge of BEC NPs decreased.
[0062] The Fourier transform infrared (FTIR) spectra of EGCG, Cu, EC NPs, BSANPs and BEC NPs are shown in Figure 2. Figure 4 As shown, the phenolic hydroxyl group in ECNPs is at 1619 cm -1 The deformation vibration peak at 1347 cm -1 The COH stretching vibration peak at 1535 cm is weaker than that of EGCG, indicating that copper ions are coupled to EGCG through phenolic hydroxyl groups. In addition, the amino group of BSA is the key to the formation of nanoparticles. The FTIR spectrum of BSANPs shows that the COH stretching vibration peak at 1535 cm is weaker than that of EGCG, indicating that copper ions are coupled to EGCG through phenolic hydroxyl groups. -1 and 1658cm -1 NH and CN vibrations can be seen in the BEC NPs. The NH and CN vibration peaks can also be observed in BEC NPs, but they are significantly weaker than those in BSANPs. It is speculated that the addition of EC NPs promotes the formation of Schiff base bonds in BSANPs.
[0063] The ultraviolet-visible-near-infrared (UV-vis-NIR) spectra of EC NPs, BSANPs and BEC NPs are shown in Figure 2. Figure 5 As shown, the absorption spectrum of BECNPs contains the characteristic absorption peak of EC NPs (275 nm), further verifying the successful synthesis of BEC NPs.
[0064] Example 2: Application of albumin nanoparticles loaded with phenolic network;
[0065] In this example, the albumin nanoparticles loaded with phenolic network prepared based on the above method are applied to drugs that promote the healing of chronic periodontitis. Through a series of experiments, its targeting and effectiveness are verified, and the fact that the encapsulation of albumin nanoparticles does not affect the effect of phenolic network in promoting the healing of chronic periodontitis is verified.
[0066] 2.1 In vitro experiments:
[0067] Cell activity assay: The effects of BEC NPs on the activity of mouse fibroblasts at concentrations of 0 μg / mL, 125 μg / mL, 250 μg / mL, 375 μg / mL, 500 μg / mL, and 625 μg / mL were tested. Figure 6As shown in the results, BEC NPs had no cytotoxicity to mouse fibroblasts at concentrations below 250 μg / mL. However, cell viability decreased significantly as the concentration increased, with relatively low cell viability rates of 77% and 80% at concentrations of 375 μg / mL and 500 μg / mL, respectively.
[0068] Live and dead staining assay: Live and dead staining assay was performed on cells treated with different BEC NPs concentrations (0 μg / mL, 125 μg / mL, 250 μg / mL, 375 μg / mL, 500 μg / mL and 625 μg / mL). The results are shown in Figure 2. Figure 7 As shown (scale bar = 100 μm). Cell death was almost negligible at concentrations below 250 μg / mL, but the number of dead cells increased significantly when the concentration increased to 375 μg / mL, indicating a potential biological risk.
[0069] ROS scavenging experiment and quantitative analysis: The in vitro antioxidant properties of EC NPs and BEC NPs were evaluated by ROS scavenging experiment. Figure 8 As shown in the figure (scale bar = 50 μm), BEC NPs can significantly scavenge ROS generated by LPS (i.e., bacterial lipopolysaccharide) stimulation, and the scavenging effect is similar to or even better than that of the EC NPs group.
[0070] Detection of mRNA expression levels of related factors by qPCR: Quantitative polymerase chain reaction (qPCR) was used to detect the messenger RNA (mRNA) expression levels of M1 and M2 phenotype-related factors in RAW 264.7 cells, such as Figure 9 As shown in the figure. LPS stimulation leads to abnormal increases in TNF-α and IL-1β levels, but BEC NPs significantly inhibit this effect, with an effect similar to or even slightly stronger than that of EC NPs. In addition, compared with the LPS group, both BEC NPs and EC NPs significantly enhanced Arg-1 expression, with comparable effects.
[0071] Flow cytometry was used to determine the cell ratio: Flow cytometry was used to determine the cell ratio of CD86+ (surface marker of M1 macrophages) and CD206+ (surface marker of M2 macrophages) in EC NPs and BEC NPs. Figure 10 The application of ECNPs and BEC NPs can reduce the proportion of M1 macrophages and increase the proportion of M2 macrophages, which can significantly improve the inflammatory state of macrophages. The effect of BEC NPs is more obvious than that of EC NPs.
[0072] Cellular uptake and inflammation targeting ability detection: The results of cellular uptake and inflammation targeting ability detection of BEC NPs are as follows Figure 11As shown (scale bar = 20 μm), the green fluorescence signal of FITC-labeled BEC NPs was located within the membrane of Dil-labeled neutrophils, demonstrating that BEC NPs had been successfully taken up into the interior of neutrophils.
[0073] Inhibition of neutrophil extracellular trap release detection: SYTOX Green staining and Annexin V-mcherry staining were used to determine the neutrophil extracellular trap (NETs) release content in the BSANPs and BEC NPs treatment groups. Figure 12 As shown in the figure (scale bar = 20 μm). The results showed that pretreatment with BSANPs had a slight inhibitory effect on PMA-induced NETs release, but the effect was not significant; while the addition of BEC NPs could effectively inhibit PMA-induced NETs release. On the other hand, the immunofluorescence staining results and quantitative analysis of neutrophil extracellular trap-related proteins MPO and NE in the NAC (ROS inhibitor) and BEC NPs treatment groups (scale bar = 20 μm) are shown in the figure. Figure 13 The results showed that both BEC NPs and NAC could effectively inhibit the release of MPO and NE from PMA-induced NETs and restrict their nuclear entry, with similar effects.
[0074] Verification of the synergistic anti-inflammatory effect: To verify the synergistic anti-inflammatory effect of BEC NPs on neutrophils and macrophages, extracted neutrophils were co-cultured with PBS, BSANPs, or BEC NPs for 0.5 h and then incubated in PMA-containing medium for 4 h. The upper PMA-containing medium was then removed, and the bottom cells and NETs were collected and inoculated into the upper chamber (0.4 μm). The lower chamber was inoculated with macrophages pre-cultured for 24 h. After 24 h of incubation, the phenotype of macrophages and the secretion of related cytokines were determined by immunofluorescence staining and PCR. In the following experiments, the control group was a macrophage group without co-culture; the model group was a macrophage group co-cultured with neutrophils and NETs; the BSANPs group was a macrophage group co-cultured with neutrophils and NETs that had pre-ingested BSANPs; and the BEC NPs group was a macrophage group co-cultured with neutrophils and NETs that had pre-ingested BEC NPs.
[0075] The immunofluorescence staining results of inflammatory-related cytokines TNF-α, IL-6, IL-10 and Arg-1 in the BSANPs and BEC NPs groups (scale bar = 50 μm) are shown in Figure 2. Figure 14As shown, the results showed that BEC NPs treatment significantly reversed the increased expression of pro-inflammatory factors (TNF-α, IL-6) caused by LPS stimulation, and promoted the expression of anti-inflammatory factors (IL-10, Arg-1), effectively improved the inflammatory state of macrophages, and promoted the polarization of macrophages to M2 type. Moreover, the anti-inflammatory effect of the BEC NPs group was better than that of the BSANPs group.
[0076] qPCR was used to detect the expression levels of messenger RNA (mRNA) of M1 and M2 phenotype-related factors in macrophages. Figure 15 As shown, the results showed that BEC NPs treatment reduced the increased expression of pro-inflammatory factors (TNF-α, IL-1β, IL-6) caused by LPS stimulation, and promoted the expression of anti-inflammatory factors (IL-10, Arg-1, TGF-β), effectively improved the inflammatory state of macrophages, and promoted the polarization of macrophages to M2 type. Moreover, the anti-inflammatory effect of the BEC NPs group was better than that of the BSANPs group.
[0077] qPCR was used to detect the expression levels of pyroptosis-related factor messenger RNA (mRNA) in macrophages in the BSA NPs and BEC NPs groups after co-culture. Figure 16 As shown, the results showed that the expression of IL-1β, IL-18, caspase1, GSDMD, and Nlrp3 in macrophages was upregulated after 4 hours of PMA stimulation, and the BEC NPs group could significantly downregulate the expression of pyroptosis-related inflammatory factors.
[0078] Determination of LDH content to detect macrophage pyroptosis Figure 17 As shown, the results showed that BEC NPs-induced pyroptosis resulted in a decrease in elevated LDH levels compared with the levels in NETs-treated macrophages, indicating that the reduction of NETs expression alleviated the pyroptosis-induced increase in LDH levels.
[0079] 2.2 In vivo experiments
[0080] Twenty-five male Wistar rats weighing 200-220 g were randomly divided into five groups (n=5) each: control, periodontitis, inflammation + BSANPs (BSANPs), inflammation + EC NPs (EC NPs), and inflammation + BEC NPs (BEC NPs). Rats were anesthetized with isoflurane and secured around the maxillary second molars with a 0.2 mm ligature. Twenty-one days later, the ligature was removed and the control and periodontitis groups received a local injection of 0.2 ml of PBS solution into the gingival sulcus, while the experimental groups received a local injection of 0.2 ml of 250 μg / mL nanoparticles. Administration was continued every other day. One week later, the rats were anesthetized as described above and euthanized by CO2 overdose. Cardiac perfusion fixation was performed with saline and 4% paraformaldehyde. Maxillary bones, hearts, livers, spleens, and kidneys were collected after fixation.
[0081] The rat maxillary tissue was fixed with 4% PFA for 2 days and scanned with microCT at 114 mA, 70 kVP, and 300 ms exposure time to observe 3D images of the rat maxillary tissue. After the scan, the maxillary tissue was demineralized and histopathological analysis (HE staining) was performed to observe the inflammation. Figure 18 As shown in the figure, the alveolar bone distance in the control group was the longest, while the alveolar bone distance in the BEC NPs group was the shortest, with a significant difference, indicating that BEC NPs have a positive effect on alveolar bone regeneration.
[0082] To further confirm the above results, HE and Masson staining were performed (eg Figure 19 ) to evaluate the inflammatory status and periodontal tissue regeneration. In the inflammation group, the periodontal pocket epithelium was separated from the enamel surface and accompanied by erosion. A large number of neutrophils, lymphocytes and plasma cells infiltrated under the epithelium, the periodontal ligament structure was disordered, and the alveolar bone height was significantly reduced. In the BSA NPs group, the epithelium at the periodontal junction was separated from the tooth surface and accompanied by a large number of inflammatory cell infiltrations. The periodontal ligament tissue was relatively disordered, the alveolar bone was absorbed, and no obvious new bone formation was observed. In the EC NPs group, the gingival morphology was close to normal; the separation of the junctional epithelium from the tooth surface was almost completely restored, the infiltration of inflammatory cells was significantly reduced, the periodontal ligament was neatly arranged, and the alveolar bone height was restored. In the BEC NPs group, the gingival morphology basically returned to normal, the junctional epithelium was close to the tooth surface, the infiltration of inflammatory cells was significantly reduced, the periodontal ligament was well arranged, and the alveolar bone height was significantly restored.
[0083] One rat was randomly selected from each group after the HE staining test, and the internal organs were removed to make tissue sections for HE staining observation. The results were as follows: Figure 20As shown in the figure, the visceral tissue structures of rats in each group were normal, indicating that BEC NPs were non-toxic to mammals and had good biocompatibility in animals.
[0084] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.
Claims
1. A method for preparing albumin nanoparticles loaded with a phenolic network, characterized in that: The following steps are involved: Step 1: Dissolve epigallocatechin gallate in anhydrous ethanol and 2+ The salt was dissolved in anhydrous ethanol, and then the Cu 2+ The ethanol solution of salt was slowly added dropwise to the ethanol solution of EGCG at a volume ratio of 3:1 and stirred to form a homogeneous solution; Step 2: The pH of the homogeneous solution was adjusted to 7.0 by dropwise addition of sodium hydroxide solution and the mixture was reacted at 37°C for 24 hours. After the reaction, the complex in the mixture was harvested by centrifugation, washed with ethanol, and finally dried under vacuum to obtain the EGCG-Cu metal phenolic network, i.e., EC NPs. Step 3: Completely dissolve bovine serum albumin in ultrapure water, and then completely dissolve EC NPs in the bovine serum albumin solution; Step 4: The aqueous solution obtained in step 3 was vigorously stirred at 25°C and anhydrous ethanol solution was injected to form a colloidal solution; Step 5: After 15 min, glutaraldehyde solution was added dropwise with continuous stirring to crosslink the molecules and form bovine serum albumin nanoparticles, i.e., BSANPs. Step 6: After 24 h of reaction, the precipitate was collected by centrifugation, washed with PBS solution, and freeze-dried for 48 h to obtain albumin nanoparticles loaded with phenolic network, namely BEC NPs.
2. The preparation method according to claim 1, characterized in that The Cu 2+ The salt is CuCl2·2H2O.
3. The preparation method according to claim 1, characterized in that The injection rate of the anhydrous ethanol solution is 2 mL / min.
4. An albumin nanoparticle loaded with a phenolic network, characterized in that The invention is prepared by the preparation method according to any one of claims 1 to 3.
5. Use of the albumin nanoparticles loaded with phenolic network according to claim 4 in the preparation of a drug for treating chronic periodontitis, characterized in that: The BEC NPs work through the following mechanisms: Specific targeting of neutrophils at sites of inflammation; EC NPs are released in response to the inflammatory microenvironment, scavenging reactive oxygen species and inhibiting the formation and release of neutrophil extracellular traps; Promote macrophage polarization to M2 type, downregulate pro-inflammatory factors TNF-α, IL-1β, IL-6 and upregulate anti-inflammatory factors IL-10, Arg-1, TGF-β; Inhibit cell pyroptosis pathway and reduce inflammatory damage; Ultimately, it can promote alveolar bone regeneration and periodontal tissue repair.
6. The use according to claim 5, characterized in that The BEC NPs were administered at a concentration of 250 μg / mL by local injection.
7. A pharmaceutical composition, characterized in that The method comprises the albumin nanoparticles loaded with phenolic network according to claim 4 and a pharmaceutically acceptable carrier, for treating chronic periodontitis.
8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition is administered by local injection.