Department of Pharmacology and Toxicology, University of Louisville School of Medicine, United States; Center for Predictive Medicine, University of Louisville, 505 S. Hancock St., Louisville, KY 40202, United States; Department of Toxicology, Forensic Medicine and Veterinary Regulations, Faculty of Veterinary Medicine, Cairo University, Egypt.
Department of Bioengineering, University of Louisville Speed School of Engineering, United States; Department of Microbiology and Immunology, University of Louisville School of Medicine, United States; Department of Pharmacology and Toxicology, University of Louisville School of Medicine, United States; Center for Predictive Medicine, University of Louisville, 505 S. Hancock St., Louisville, KY 40202, United States.
J Control Release. 2019 Mar 10;297:3-13. doi: 10.1016/j.jconrel.2019.01.036. Epub 2019 Jan 25.
The interaction of the periodontal pathogen Porphyromonas gingivalis (Pg) with commensal streptococci promotes Pg colonization of the oral cavity. Previously, we demonstrated that a peptide (BAR) derived from Streptococcus gordonii (Sg) potently inhibited adherence of Pg to streptococci and reduced Pg virulence in a mouse model of periodontitis. Thus, BAR may represent a novel therapeutic to control periodontitis by preventing Pg colonization of the oral cavity. However, while BAR inhibited the initial formation of Pg/Sg biofilms, much higher concentrations of peptide were required to disrupt an established Pg/Sg biofilm. To improve the activity of the peptide, poly(lactic-co-glycolic acid) (PLGA) nanoparticles were surface-modified with BAR and shown to more potently disrupt Pg/Sg biofilms relative to an equimolar amount of free peptide. The goal of this work was to determine the in vivo efficacy of BAR-modified NPs (BNPs) and to assess the toxicity of BNPs against human gingival epithelial cells. In vivo efficacy of BNPs was assessed using a murine model of periodontitis by measuring alveolar bone resorption and gingival IL-17 expression as outcomes of Pg-induced inflammation. Infection of mice with Pg and Sg resulted in a significant increase in alveolar bone loss and gingival IL-17 expression over sham-infected animals. Treatment of Pg/Sg infected mice with BNPs reduced bone loss and IL-17 expression almost to the levels of sham-infected mice and to a greater extent than treatment with an equimolar amount of free BAR. The cytotoxicity of the maximum concentration of BNPs and free BAR used in in vitro and in vivo studies (1.3 and 3.4 μM), was evaluated in telomerase immortalized gingival keratinocytes (TIGKs) by measuring cell viability, cell lysis and apoptosis. BNPs were also tested for hemolytic activity against sheep erythrocytes. TIGKs treated with BNPs or free BAR demonstrated >90% viability and no significant lysis or apoptosis relative to untreated cells. In addition, neither BNPs nor free BAR exhibited hemolytic activity. In summary, BNPs were non-toxic within the evaluated concentration range of 1.3-3.4 μM and provided more efficacious protection against Pg-induced inflammation in vivo, highlighting the potential of BNPs as a biocompatible platform for translatable oral biofilm applications.
牙周病原体牙龈卟啉单胞菌（Pg）与共生链球菌的相互作用促进了 Pg 在口腔中的定植。此前，我们证明了来源于链球菌（Sg）的一种肽（BAR）可有效抑制 Pg 与链球菌的黏附，并在牙周炎的小鼠模型中降低 Pg 的毒力。因此，BAR 可能代表一种通过防止 Pg 定植口腔来控制牙周炎的新型治疗方法。然而，尽管 BAR 抑制了 Pg/Sg 生物膜的初始形成，但需要更高浓度的肽才能破坏已建立的 Pg/Sg 生物膜。为了提高肽的活性，将聚（乳酸-共-乙醇酸）（PLGA）纳米颗粒用 BAR 表面修饰，并显示出比等量游离肽更有效地破坏 Pg/Sg 生物膜。这项工作的目的是确定 BAR 修饰的纳米颗粒（BNPs）的体内疗效，并评估 BNPs 对人牙龈上皮细胞的毒性。通过测量牙槽骨吸收和牙龈白细胞介素-17（IL-17）表达作为 Pg 诱导的炎症的结果，使用牙周炎的小鼠模型评估 BNPs 的体内疗效。Pg 和 Sg 感染小鼠导致牙槽骨丢失和牙龈 IL-17 表达显著增加，超过假感染动物。用 BNPs 治疗 Pg/Sg 感染的小鼠可使骨丢失和 IL-17 表达减少到与假感染小鼠相似的水平，并且比用等量游离 BAR 治疗的效果更显著。在体外和体内研究中使用的 BNPs 和游离 BAR 的最大浓度（1.3 和 3.4 μM）的细胞毒性，通过测量细胞活力、细胞裂解和细胞凋亡，在端粒酶永生化的牙龈角质形成细胞（TIGKs）中进行评估。还测试了 BNPs 对绵羊红细胞的溶血活性。与未处理的细胞相比，用 BNPs 或游离 BAR 处理的 TIGKs 的活力>90%，且没有明显的裂解或凋亡。此外，BNPs 和游离 BAR 均没有溶血活性。总之，BNPs 在 1.3-3.4 μM 的评估浓度范围内无毒性，并且在体内对 Pg 诱导的炎症提供了更有效的保护，突出了 BNPs 作为可转化口腔生物膜应用的生物相容性平台的潜力。
