Preparation And Anti-inflammatory Effects Of Nanoparticles Loaded With PMX-53

Background:As is known to all, periodontitis is a chronic inflammatory disease, which is characterized by destruction of periodontal tissue and resorption of alveolar bone and the incidence rate as high as 80%. It not only destroy periodontal tissue and block chewing function, but also induce systemic disease:diabetes,cardiovascular disease,Alzheimer’s and respiratory diseases, etc. Thus, hancing prevention and treatment of periodontitis will have important significance to improve quality of life and maintain the health. Although basic treatment of periodontitis is the primary methods, local adjuvant drug therapies are the improtant means in the clinical treatment of periodontitis. Currently, main medicine of treating periodontitis is imported minocycline hydrochloride tetracycline broad-spectrum antibiotics, but this drug has many disadvantages such as easy to produce drug resistance and making flora imbalance in the periodontium, so that it is used limitly in the clinical application.Therefore, it is significant to develop a drug which has small side effects and independent intellectual property rights to treat periodontitis. PMX-53 that is a polypeptide of synthesis simulaties chemical structure of C5 a R as a C5 a R antagonist.Gingipains are one of the major virulence factors of Porphyromonas gingivals(Pg). They have C5 convertase activity which can degrade C5 into C5 a to activate C5a-C5 a R signaling pathway to make periodontal immune inflammation. PMX-53 can target to block the C5a-C5 a R signaling pathway to develop anti-inflammatory effects. Hajishengallis’ s studies have found that PMX-53 has more obvious anti-inflammatory effects when it was applied to mice periodontal model. In addition,PMX-53 also could reduce the destruction of the alveolar bone. However, the duration of drug action is very short, so it needs be injected frequently and multi-points injection are painful and inconvent to the patients. Gelatin as a natural polymeric material has been widely used as a drug carriersin in the clinic, which isV biocompatible, non-toxic, non-antigenic, etc. In conclusion, the experiment uesd gelatin nanoparticles loaded with PMX-53 that have great anti-inflammatory activity and biological safety. Then we observed anti-inflammatory effects in vivo and vitro,to lay the foundation for its clinical application.Methods:1. Preparation of gelatin nanoparticles loaded with PMX-53Gelatin nanoparticles loaded with PMX-53 were prepared by two-step desolvation processes, then were characterized.2. Biological safety evaluation of drug-loaded nanoparticles.RAW264.7 cells of the number growth period were cultured in sterile 96 well plates. The experiments were divided into a blank control group and various concentration of drug-loaded nanoparticles groups(0.025%, 0.05%, 0.1%, 0.2%,0.4%, 0.8%), then cytotoxic of drug-loaded nanoparticles were tested by MTT at 24 h,48h, 72 h.3.The anti-inflammatory effects of drug-loaded nanoparticles in vitroRAW264.7 cells were cocultured with gingipains to simulate inflammatory environment in vitro. The experiments were divided into 4 groups: a control group, a gingipains group, a gingipains+PMX-53(1μg/ml) group, and a gingipains+nanoparticles loaded with PMX-53(4.17mg/ml) group. Then levels of IL-1β, IL-6 and TNF-α were detected by the ELISA test, in order to detect the anti-inflammatory effects of drug-loaded nanoparticles.4. The anti-inflammatory effects of drug-loaded nanoparticles in vivoThe experiments used 24-month-old male Bama minipigs, and selected the fourth premolar and the first molar tooth as experimental tooth. The model of periodontitis minipigs were established with Pg(1×109) and silk(or steel wire)ligation. When models were established successfully, the experiments were divided into 4 groups randomly:a negative control group(simple periodontitis group), a gelatin nanoparticles group, a drug-loaded particles group, a positive control group(periocline). Then we detected the anti-inflammatory effects of drug-loaded nanoparticles in vivo.Results:1. The nanoparticles loaded with PMX-53 looked faint yellow.SEM revealed that nanoparticles loaded with PMX-53 were spherical and 51.24% of them were 40~60nm in diameter. Drug content was 0.24μg/mg. The encapsulation rate was 46.2%. The drug release time can reach 7days.2. MTT resultsMTT results showed that cytotoxicity of 0.025%, 0.05%, 0.1%, 0.2% and 0.4%leaching liquor groups were grade 1, within safe limits. 0.8% leaching liquor group gradually showed low cytotoxicity with increasing days of cell culture.3. Anti-inflammatory effects in vitroELISA results showed that: The levels of IL-1β and IL-6 of gingipains groups were higher than those of the control group after 48 hours(P<0.05). The levels of IL-1β and IL-6 of gingipains + nanoparticles loaded with PMX-53 group and gingipains + PMX-53 group were lower than gingipains group(P<0.05). Furthermore,there was a significant reduction in the levels of IL-6 of gingipains+nanoparticles loaded with PMX-53 group(P<0.01).4. Anti-inflammatory effects on vivo4.1 Gingivals of minipigs were congestive, swollen and puniceous by naked eyesobservation at the 7th week establishing models. Inflammatiory gingivals weresignificantly bleeding with probing. Afer 4 weeks with drug therapies,inflammation of each experimental groups were relieved.4.2 Probing examination showed that each experimental groups were improvedsignificantly(P<0.01).Furthermore, the drug-loaded nanoparticles group was mostobvious(P <0.01).4.3 X-ray showed that each experimental group was significant absorption ofalveolar bone at the 7th week after modeling. Afer 4 weeks with drug therapies, thevdensity and height of bone increased most obvious between the gelatinnanoparticles group and the drug-loaded nanoparticles group. Density and height ofbone of the periocline group were similar with the negative control group.Conclusions:1. We successfully made the nanoparticles loaded with PMX-53. Processes of preparation were simple and feasible.2. Nanoparticles loaded with PMX-53 were low cytotoxicity and good biocompatibility.3. Nanoparticles loaded with PMX-53 can significantly inhibit the levels of IL-1β and IL-6 in vitro. It was proved that nanoparticles loaded with PMX-53 had obviously anti-inflammatory effects.4. We successfully established model of periodontitis in minipigs. And nanoparticles loaded with PMX-53 had good anti-inflammatory effects on periodontitis of minipigs.