BACKGROUND:Staphylococcal infections and its biofilm formation can occur when orthopedic implants or wound is healing, and are regulated by bacterial population sensing mechanism. RNAIII inhibiting peptide intervenes the quorum-sensing system of staphylococcal and blocks the signal transduction among staphylococcal cells, and inhibits staphylococcal biofilm formation, and then prevents staphylococcal infections.
OBJECTIVE:To investigate the influence of RNAIII inhibiting peptide on the adhesion of staphylococcus epidermis to the Hela cells.
METHODS:The Hela cells were cultured in vitro. There were four groups in this study. In the blank group, saline with dimethyl sulfoxide was added in each wel . In the RNAIII inhibiting peptide group, dimethyl sulfoxide solution containing RNAIII inhibiting peptide was added. In the levofloxacin group, levofloxacin was added. In the combination group, the dose was in accordance with above methods. Using intergroup control method, the adhesion of staphylococcus epidermis to the Hela cells was compared under the effects of saline, RNAIII inhibiting peptide and levofloxacin and their combination.
RESULTS AND CONCLUSION:In the blank group, abundant bacterial adhered to Hela cells. The number of adhered bacteria was significantly lower in each medicine group than in the blank group (P<0.001). The spot count was significantly lower in the levofloxacin group than in the RNAIII inhibiting peptide group (P<0.05). In the combination group, the number of bacteria adhered to Hela cells was decreased (P<0.01). Results verified that RNAIII inhibiting peptide effectively suppressed the adhesion of staphylococcus epidermis to the host cells, and showed synergistic effects on antibiotics.

OBJECTIVE: To evaluate the histocompatibility of poly (lactic-co-glycolic acid)/RNA Ⅲ inhibiting peptide (PLGNRIP) sustained release microspheres.METHODS: The crude peptide comprising N to C-terminals was synthesized using Fmoc method. The crude synthetic RNAⅢ peptide was purified by reverse phase high performance liquid chromatography, followed by component harvesting according to ultraviolet absorption peak, and freeze-drying. PLGNRIP sustained release microspheres with a diameter of 50-70 pm were prepared using liquid-phase multiple emulsion method. The histocompatibility of PLGNRIP sustained release microscopes were preliminarily evaluated through the use of acute general toxicity test, MTT cytotoxicity test, intramuscular implantation test, sensitivity test, and pyrogen test.RESULTS: Acute general toxicity test results showed that all included animals survived and presented with no toxicosis reaction and obviously changed body mass. MTT cytotoxicity test results revealed that the average relative growth rate of cells from two eluents was over 85%, with cytotoxicity grade 1, which indicated no cytotoxicity. Intramuscular implantation tests showed that at 4 weeks after implantation of RiP powder or PLGNRIP microscopes, no obviously congested, degenerated, or necrotic tissue was observed. All RIP powder and a part PLGNRIP microscopes were degraded. Fibroblasts accounted for a large proportion in all cells. NO inflammatory cell infiltration, involving neutrophits and multinucleated giant celts, was observed. Sensitivity test rasults displayed that the average primary irritation index was 0.38, 0.33, arid 0.31 in the eluent stock solution, 2% dinitoflruorobenzene, and physiological saline-administerd groups, respectively. Pyrogen test results showed that fervescence of each rabbit in the experiment was under 0.5 ℃ and the sum of fervescence was under 1.3 ℃ .This is in coincidence with evaluation criteria of pyrogen test.CONCLUSION: PLGNRIP sustained release microspheras exhibit good histocompatibility.

OBJECTIVE: To evaluate blood compatibility of polyaiticglycolic acid/RNA Ⅲ inhibiting peptide (PLGA/RIP) delayed release microspheres.METHODS: ① Preparation of PLGA/RIP microspheres: The solid-phase synthesis (Fmoc) method was used to synthesize RIP crude sample from C end to N end; the synthesized crude peptide was purified by the reverse phase high performance liquid chromatography. According to UV absorption peak, the components were collected and freeze-dried, to obtain RIP purifications. Then liquid-phase multiple emulsion method was used to prepare PLGA/RIP microspheres at the diameter of 50-70 μm. ② Preparation of eluent: The PLGA/RIP microsphere powders were eluted with sterile physiological saline at 37 ℃, to prepare 1 g/L eluent; then 0.5 g/L eluent was obtained adding equal volume of sterile physiological saline. The hemolysis test, blood clotting test, and platelet aggregation test were conducted to measure prothrombin time and activated partial thromboplastin time, to observe the influence on rabbit leucocytes, erythrocytes and thrombocytes, and to preliminarily evaluate the blood compatibility of PLGA/RIP microspheres. RESULTS: ①The haemolysis rates of eluent stock solution and 0.5 g/L eluents were 3.24% and 2.67% respectively, which were in coincidence with the criteria of medical biomaterials, less than 5%. ② The eluent stock solution and 0.5 g/L eluents of PLGA/RIP microspheres had no significant effect on rabbit clotting time, prothrombin time and activated partial thromboplastin time, the number of rabbit leucocytes, erythrocytes and thrombocytes, as well as platelet aggregation.CONCLUSION: PLGA/RIP delayed release microspheres have a good blood compatibility.

BACKGROUND: Staphylococci inhibitor, RNAⅢ inhibiting peptide, has been firstly synthesized in China, while RNAIII inhibiting peptide/bioinspired phosphorylcholine-based cytomembrane coating (RIP/PC) has been also prepared.OBJECTIVE: To evaluate the blood compatibility of RIP/PC.DESIGN, TIME AND SETTING: A randomized controlled animal experiment was performed at Institute of Clinical Pharmacologic Research and Medical Animal Experimental Center of General Hospital of Chinese PLA from October 2005 to October 2007.MATERIALS: A total of 30 healthy adult New Zealand rabbits were provided by Animal Experimental Center of General Hospital of Chinese PLA.METHODS: The 316L stainless steel was dip into a mixture of 5 g/L quaternionic copolymer tetrahydrpfuran and 10% RNAⅢ inhibiting peptide to obtain stable polymer coating, which was extracted at density of 1 cm~2/mL in saline at 37 ℃ for 72 hours to get 100% eluent. The same volume of saline was added to make 50% eluent. The effects of RIP/PC on leukocytes, eryfhrocytes,and blood platelet of rabbits were detected via measuring prothrombin time and activated thrombin time during hemolysis test,hemagglutinatin test, and blood platelet aggregation test.MAIN OUTCOME MEASURES: Hemolytic ratio of erythrocytes, clotting time, prothrombin time, activated thrombin time, amounts of leukocytes, erythrocytes, and blood platelet, as well as blood platelet aggregation.RESULTS: Hemolysis test showed that the hemolytic ratio was 3.08% and 1.88% of 100% and 50% eluent, respectively; both the hemolytic ratios were < 5%, suggesting being coincidence with hemolytic requests of biological materials. The hemagglutinatin test showed that both 100% and 50% eluent did not have any effect on clotting time, prothrombin time, activated thrombin time,amounts of leukocytes, erythrocytes, and blood platelet, as well as blood platelet aggregation.CONCLUSION: The firstly synthesized RIP/PC has a good biocompatibility.

OBJECTIVE:To evaluate the histocompatibility of poly(lactic-co-glycolic acid)/RNA Ⅲ inhibiting peptide(PLGA/RIP)sustained release microspheres.METHODS:The crude peptide comprising N to C-terminals was synthesized using Fmoc method.The crude synthetic RNAIII peptide was purified by reverse phase high performance liquid chromatography,followed by component harvesting according to ultraviolet absorption peak,and freeze-drying.PLGA/RIP sustained release microspheres with a diameter of 50-70?m were prepared using liquid-phase multiple emulsion method.The histocompatibility of PLGA/RIP sustained release microscopes were preliminarily evaluated through the use of acute general toxicity test,MTT cytotoxicity test,intramuscular implantation test,sensitivity test,and pyrogen test.RESULTS:Acute general toxicity test results showed that all included animals survived and presented with no toxicosis reaction and obviously changed body mass.MTT cytotoxicity test results revealed that the average relative growth rate of cells from two eluents was over 85%,with cytotoxicity grade 1,which indicates no cytotoxicity.Intramuscular implantation tests showed that at 4 weeks after implantation of RIP powder or PLGA/RIP microscopes,no obviously congested,degenerated,or necrotic tissue was observed.All RIP powder and a part PLGA/RIP microscopes were degraded.Fibroblasts accounted for a large proportion in all cells.No inflammatory cell infiltration,involving neutrophils and multinucleated giant cells,was observed.Sensitivity test results displayed that the average primary irritation index was 0.38,0.33,and 0.31 in the eluent stock solution,2% dinitoflruorobenzene,and physiological saline-administerd groups,respectively.Pyrogen test results showed that fervescence of each rabbit in the experiment was under 0.5℃ and the sum of fervescence was under 1.3℃.This is in coincidence with evaluation criteria of pyrogen test.CONCLUSION:PLGA/RIP sustained release microspheres exhibit good histocompatibility.