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. 2024 Apr 15;22(1):181.
doi: 10.1186/s12951-024-02461-0.

The application of phenylboronic acid pinacol ester functionalized ROS-responsive multifunctional nanoparticles in the treatment of Periodontitis

Jinhong Chen #  1 , Aihua Luo #  1 , Mengmeng Xu  1 , Yao Zhang  1 , Zheng Wang  1 , Shuang Yu  1 , Li Zhu  2 , Wei Wu  3 , Deqin Yang  4
Affiliations

The application of phenylboronic acid pinacol ester functionalized ROS-responsive multifunctional nanoparticles in the treatment of Periodontitis

Jinhong Chen et al. J Nanobiotechnology. .

Abstract

Periodontitis is an inflammatory disease induced by the complex interactions between the host immune system and the microbiota of dental plaque. Oxidative stress and the inflammatory microenvironment resulting from periodontitis are among the primary factors contributing to the progression of the disease. Additionally, the presence of dental plaque microbiota plays a significant role in affecting the condition. Consequently, treatment strategies for periodontitis should be multi-faceted. In this study, a reactive oxygen species (ROS)-responsive drug delivery system was developed by structurally modifying hyaluronic acid (HA) with phenylboronic acid pinacol ester (PBAP). Curcumin (CUR) was encapsulated in this drug delivery system to form curcumin-loaded nanoparticles (HA@CUR NPs). The release results indicate that CUR can be rapidly released in a ROS environment to reach the concentration required for treatment. In terms of uptake, HA can effectively enhance cellular uptake of NPs because it specifically recognizes CD44 expressed by normal cells. Moreover, HA@CUR NPs not only retained the antimicrobial efficacy of CUR, but also exhibited more pronounced anti-inflammatory and anti-oxidative stress functions both in vivo and in vitro. This provides a good potential drug delivery system for the treatment of periodontitis, and could offer valuable insights for dental therapeutics targeting periodontal diseases.

Keywords: Curcumin; Hyaluronic acid; Periodontitis; Phenylboronic acid pinacol ester; ROS-responsive.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Scheme 1
Scheme 1
The application of HA@CUR NPs as ROS-responsive multifunctional nanoparticles in the treatment of periodontitis, and an explanation of their potential mechanism for alleviating chronic inflammation in an SD rat model
Fig. 1
Fig. 1
Preparation and Characterization of HA@CUR NPs. (A) Illustrative schematic of the preparation process for HA@CUR NPs; (B) TEM images depicting the morphology and aqueous particle size distribution of HA@CUR NPs (Bar = 1 μm); (C) Variation in particle size of HA@CUR NPs over the course of 1, 3, 5, and 7 days of storage at room temperature, and (D) changes in the dispersity coefficient; (E) TEM images of HA@CUR NPs before and after the addition of H2O2. (Bar = 1 μm); (F) Release rate profiles of HA@CUR NPs in the different concentrations of H2O2. (n.s.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001)
Fig. 2
Fig. 2
(A) Fluorescent microscopic observation (blue: Hoechst, red: Cy5.5) of RAW 264.7, HGFs and Caco2 uptake of HA-Cy5.5 NPs before and after treatment with anti CD44 and HA; (B) FCM and (C) quantitative analysis of RAW264.7 cell uptake of RAW 264.7, HGFs and Caco2 uptake of HA-Cy5.5 NPs before and after treatment with anti CD44 and HA. (n.s.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001)
Fig. 3
Fig. 3
(A) Fluorescent microscopic observation (blue: Hoechst, red: Cy5.5) for non-activated RAW264.7 and activated RAW 264.7 uptake of HA-Cy5.5 NPs before and after treatment with anti CD44 and HA. (B) FCM and (C) quantitative analysis for non-activated RAW 264.7 and activated RAW 264.7 uptake of HA-Cy5.5 NPs before and after treatment with anti CD44 and HA. (n.s.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001)
Fig. 4
Fig. 4
Inhibitory and bactericidal effects of HA@CUR NPs on P.g.; (A) Inhibition rate of P.g. by different concentrations of CUR solution and HA@CUR NPs; (B) Live/Dead staining of P.g. biofilms following the addition of HA@CUR NPs and CUR at equivalent drug concentrations; (C) Representative scanning electron microscopy images of P.g. following treatment with PBS, HA@CUR NPs, and CUR at the same drug concentrations; (D) Colony formation experiments of P.g. after treatment with PBS, HA@CUR NPs, and CUR at equivalent drug concentrations
Fig. 5
Fig. 5
In vitro antioxidant capacity of HA@CUR NPs. (A) The free radical scavenging activity of HA@CUR NPs and CUR against DPPH and (B) ABTS radicals, and (C) the iron reduction ability assessment. (D, E) The capacity of equivalent concentrations of HA@CUR NPs and CUR to clear intracellular ROS as observed under laser confocal microscopy and their quantitative analysis. (F) FCM detection of ROS clearance in RAW 264.7 cells treated with H2O2 by HA@CUR NPs and CUR, and (G) the quantification of fluorescence intensity. (H) Protective effects of HA@CUR NPs and CUR on RAW264.7 cells stimulated with a solution of 1600 µM concentration of H2O2, where both increased cell viability. (I-K) HA@CUR NPs and CUR enhance the gene expression of antioxidative factors HO-1, SOD, and CAT. (n = 3) (n.s.: not significant; *p < 0.05; **p < 0.01, and ***p < 0.001)
Fig. 6
Fig. 6
In vitro study of the effects of HA@CUR NPs and CUR on RAW 264.7 cell phenotypic transformation and gene expression of inflammatory factors under LPS stimulation. (A) FCM was used to assess the influence of HA@CUR NPs and CUR on the ratio of M1 to M2 macrophage phenotypes after LPS stimulation. CD86 (red, in the PE channel) and CD206 (red, in the PE channel) were used to specifically mark M1 and M2 type macrophages, respectively (antibody F4/80 labeled all macrophages, green, in the FITC channel). qPCR results of M1 macrophage marker gene (B) Arg1 and M2 macrophage marker gene (C) iNOS expression after RAW 264.7 cells were pre-treated with HA@CUR NPs and free CUR. (D) The gene expression levels of inflammatory factors (TNF-α, IL-1β, IL-6, Mmp8, COX-2). (n = 3) (n.s.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001)
Fig. 7
Fig. 7
(A) Schematic of the rat periodontitis treatment process. (B) Three-dimensional reconstructed images and two-dimensional sagittal plane X-ray images of maxillary alveolar bone on the buccal and palatal sides under different experimental conditions; scale bar: 1.0 mm. Mean values of bone quality parameters for each treatment group: (C) CEJ-ABC, (D) BV/TV, (E) Tb.N, (F) Tb.Th, and (G) Tb.Sp. (n = 3) (n.s.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001)
Fig. 8
Fig. 8
(A, B) The ability of HA@CUR NPs to scavenge ROS was observed by fluorescence imaging using an in vivo imaging system as a test device, which then yielded relative semi-quantitative information. (C-H) RNA expression levels of bacterial virulence factors in periodontal tissues (n = 3). (n.s.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001)
Fig. 9
Fig. 9
(A) H&E and (B) TRAP staining in the interdental area of the maxillary first and second molars.Note: Black arrows in the images indicate osteoclasts (cells positive for TRAP staining with three or more nuclei). (C) Quantitative measurements of osteoclast numbers on the alveolar bone surface in the region of the maxillary first and second molars. (n = 3) (n.s.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001)
Fig. 10
Fig. 10
(A) Representative immunohistochemical staining images of TNF-α, IL-1β, and IL-6 in the maxillary bone after treatment. Quantitative analysis of staining intensity for (B) TNF-α, (C) IL-1β, and (D) IL-6. RNA expression levels of (E) TNF-α, (F) IL-1β, and (G) IL-6 in periodontal tissues (n = 3). (n.s.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001)
Fig. 11
Fig. 11
Representative immunohistochemical staining images of HO-1, SOD, and CAT in the maxilla following treatment (A) Quantitative analysis of staining intensity for (B) HO-1, (C) SOD, and (D) CAT. RNA expression levels of (E) HO-1, (F) SOD, and (G) CAT in periodontal tissues (n = 3). (n.s.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001)
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