Self-assembly Responsive Nanocarrier-Mediated Dual Drug Delivery System To Treat Periodontitis In Diabetic Rats

Diabetes mellitus aggravates the risk and severity of periodontitis.The precise and controlled drug delivery to treat periodontitis in patients with diabetes remains a significant clinical challenge.Nano-drug delivery systems have the advantages of improving drug utilization,reducing drug side effects,improving drug solubility,reducing drug toxicity,and increasing drug stability.They have great clinical application value in the treatment of diabetes,cancer,and infectious diseases.However,the complex microenvironment of diabetes has higher requirements on the drug loading efficiency and the effect of nano-drug delivery systems.Firstly,hyperglycemia status and resultant advanced glycation end products(AGEs)formation could induce reactive oxygen species(ROS)accumulation,exacerbate inflammation,break the balance of osteoclasts and osteoblasts,lead to bone metabolism disorders,resulting in more rapid and severe destruction of periodontal bone.Secondly,prolonged exposure of bacteria to high level of ROS induces bacterial resistance and reduces the antibacterial efficacy of nano-drug delivery systems.Finally,glucose metabolism disorders can easily lead to secondary immune response defects,which will cause corresponding changes in the periodontal microecological environment,and increase the secretion of pathogenic factors.At the same time,bacterial metabolism will make the biofilm acidic,forming a special biofilm microenvironment.Therefore,it is of great significance to develop a precise and controlled drug delivery strategy for the microenvironment to treat diabetic periodontitis.Objectives:To design a novel self-assembled,dual responsive,and dual drug-loading nanocarrier system,which comprised two parts:the hydrophobiclipidcoreformedby1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethyleneglycol)(DSPE-PEG)loaded with alpha-lipoic acid capable of scavenging ROS,and a hydrophilic shell comprising a poly(amidoamine)dendrimer(PAMAM)encapsulated with broad-spectrum antibacterial minocycline hydrochloride.To explore the biological effects of the self-assembled responsive dual drug-loaded nanoparticles on periodontitis in diabetic rats.Methods:The pH-sensitive PAMAM and the lipase-sensitive DSPE-PEG were covalently linked,minocycline hydrochloride was loaded in PAMAM internal cavity,andα-lipoic acid was loaded by nano-coprecipitation.The final product pH/lipase responsive dual drug-loading nanoparticles were detected by ~1H Nuclear Magnetic Resonance spectroscopy(~1H NMR),dynamic light scattering(DLS),Fourier Transform Infrared spectroscopy(FTIR),transmission electron microscope(TEM)to characterize the physical and chemical properties such as morphology,structure,particle size,zeta potential,drug loading performance,as well as pH/lipase responsiveness.The in vitro drug release behavior of the nanomaterials was explored by dialysis method.The cytotoxicity of the nanomaterials was detected by cell counting test(Cell Counting Kit-8,CCK8).Using rhodamine B,nile red and fluorescein isothiocyanate isomer(FITC)as fluorescent probes,the cell uptake behaviors and the transport pathways of nanomaterials in cells were detected by flow cytometry and confocal microscopy.Bone marrow mesenchymal stem cells(BMSCs)were cultured with AGEs to mimick diabetes microenvironment in vitro,and the osteogenic effects of the nanomaterials on BMSCs were investigated by alizarin red staining and Quantitative Real-time Polymerase Chain Reaction(q RT-PCR).The effects of the nanomaterials on the levels of intracellular ROS and inflammatory factor inducible nitric oxide synthase(i NOS)were detected by reactive oxygen species kit and immunofluorescence staining.The antibacterial activities of the nanomaterials against planktonic bacteria and bacterial biofilms were detected in vitro by minimum inhibitory concentration(MIC),live/dead staining of biofilms,scanning electron microscope(SEM).A model of diabetic periodontitis rats was established,and the inhibitory effects of the nanomaterials on alveolar resorption of periodontitis in diabetic rats were evaluated by Micro-CT,Hematoxylin and Eosin(H&E)staining and Masson staining,and the antibacterial effects on the plaque biofilm of diabetic periodontitis rats were detected by coating plate method and SEM.Results:The nanomaterials were about 12 nm in size,positively charged on the surface,round particles,and the drug loading content ofα-lipoic acid and minocycline were 13.61%and 17.17%,respectively.Drug release from nanomaterial was enhanced in an acidic and lipase-containing environment.The nanomaterials had good biocompatibility and were effectively endocytosed by BMSCs.Confocal microscopy observed that the nanomaterials were mainly distributed in the cytoplasm and lysosomes after entering cells.The nanomaterials relieved AGEs-suppressed osteogenic differentiation of BMSCs and alleviated AGEs-induced ROS production and inflammation in vitro.Besides,the nanomaterials had good bacteriostatic activity and anti-biofilm activity against planktonic bacteria and bacterial biofilms in vitro.The results of in vivo experiments show that the nanomaterials could promote the remodeling of periodontal tissue and the attachment of periodontal ligament,inhibit the formation of dental plaque biofilm,and suppress periodontal bone loss in rats with diabetic periodontitis.Conclusions:This novel pH/lipase dual-responsive dual drug-loading nanosystem was designed to effectively carry the antioxidant/α-lipoic acid and the antibacterial minocycline,at the same time to realize the intelligent and responsive drug release,resulting in inhibiting periodontal bone loss in diabetic rats.This effective and versatile drug-delivery strategy has good potential applications and provides a new research idea for the clinical treatment of periodontitis in diabetic patients.