Preparation And Biological Evaluation Of Two Novel Piperazine Polymer With Antibacterial Properties

For humans, the bacterial has two sides. In order to fight with the bacterial, antibiotics are always used. However, antibiotic resistance has increasingly become an uncontrollable problem in recent years. As a result, development of novel antibacterial materials which are not rely on traditional antibiotics is of great significance. In this paper, we compare the inorganic antibacterial materials and organic antibacterial materials existing currently, and finally choose the antibacterial polymer as the research object. In my present, a novel piperazine polymer(PE) with unique structures was synthesized based on piperazine(PA) and ethylenediaminetetraacetic dianhydride(EDTAD). Firstly, Fourier transform infrared spectrometry(FTIR), Nuclear magnetic resonance spectrometer(NMR), Elementary analysis(EA), Differential scannning calorimetry(DSC), Thermal Gravity Analysis(TG) and Optical microscope were used to characterize its basic structure and performance. Then, we test the antibacterial properties of PE against gram positive bacteria(S.aureus) and gram negative bacteria(E.coli) and carried out a preliminary study on their mechanism of action. Thereafter, the cytotoxicity of PE was evaluated using SD rats calvaria osteoblasts. After that, the mechano-growth factor MGF and its 24 amino acid peptide analog corresponding to the unique C-terminal E-domain(MGF-Ct24E) was introduced into the PE using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide(EDC) and N-hydroxysuccinimide(NHS), and the obtained polymer(PEM) was expected to have a lower cytotoxicity. The basic structure and performance of PEM were characterized using FTIR, NMR, EA, amino acid analyzer(AAA), DSC and TG at first. Then, the antibacterial properties and cytotoxicity of PEM were tested respectively. Finally, the two kinds of polymer were compound onto the surface of porous hydroxyapatite(HA) ceramic, which is well known for its good biocompatibility and bone conductivity. After that, the antibacterial properties and biocompatibility of the obtained compounds were evaluated immediately. The main works and conclusions are included as follows:① To prepared a novel piperazine polymer PE through a simple and green way, we chosen PA and EDTAD as raw materials:1) FTIR, 13 C NMR and EA analysis showed that PE was prepared successfully, and the molar ratio of PA and EDTAD in PE was about 1:1.2) The glass transition temperature(Tg) and decomposition temperature of the synthetic polymer PE were 50.09℃ and 200℃, respectively.3) PE formed a gel of a diameter about 5μm at low temperatures, and as the temperature increases, it dissociated.②The antibacterial properties of PE against S.aureus and E.coli were characterized by susceptibility test, Minimal inhibitory concentration(MIC) and Minimal bactericidal concentration(MBC) test, growth inhibitory test, respectively. And the detections related to the biofilm formation were also did. To better understand the antibacterial properties of PE, we investigated the influence of culture conditions on MIC.1) Compared with ciprofloxacin, PE could inhibit and kill the two kinds of bacterial, especially the E.coli more significantly.2) The antibacterial properties of PE was affected by the environment pH and initial bacteria inoculation quantity. As the environment pH value rise, the MIC value of PE to bacteria present the downward trend after rising first. As the amount of initial bacteria inoculated rise, the MIC value of PE to bacteria increased as well.3) Compared with ciprofloxacin, PE could kill planktonic and adhering bacteria in a very rapid manner, could inhibit the bioflim formation of E.coli and S.aureus, but had little effect on the bacterial inside the biofilm.③ To understand the antibacterial mechanism of PE, a certain concentration of PE was acting on E.coli and S.aureus for a period of time. The changes of the surface morphology and ultrastructure of E.coli and S.aureus were observed by scanning electron microscopy(SEM) and transmission electron microscopy(TEM). The structure of bacteria cell wall were characterized using FTIR and UV. The integrity of bacteria cell membrane were investigated by determining the leakage of K+, Mg2+, LDH and β-galactosidase. The content change and damage of bacteria DNA were also tested to understand the effect of PE on bacteria genetic.1) PE could disrupt both the cell wall and cell membrane of bacteria, and led the leakage of K+, Mg2+, LDH, β-galactosidase and DNA.2) PE could break the DNA of bacteria, and ultimately affect their proliferations.④ In this study, we evaluated the cytotoxicity of PE. The rat calvarial osteoblasts were chosen as the test cell type, and the cell spreading, proliferation, cycle, differentiation and mineralization were assessed.1) PE almost wouldn’t affect the cell spreading in 4h co-culture, and with extended to 24 h, the influence of PE on osteoblasts spreading exhibited a concentrationdependent manner, but all was better than that of ciprofloxacin.2) PE showed a concentration-dependently inhibitory effect on rat calvarial osteoblasts proliferation, and its main functions include:(a) when the concentration of PE reached a certain value, it would affect the osteoblast spreading, or reduce its number;(b) PE could hinder the osteoblasts into the S phase, accumulate in the G1 phase, and finally leading to G2/M phase arrest.3) Compared with ciprofloxacin, PE showed a lower inhibitory effect on rat calvarial osteoblasts differentiation and mineralization, and the inhibitory effect depended on the concentration of PE.⑤ MGF-Ct24 E was introduced into PE via a stable covalent amide bond by using EDC and NHS as the condensing agent, the basic structure and performance of obtained polymer PEM were shown as follow.1) FTIR, 13 C NMR, EA and AAA analysis showed that MGF-Ct24 E was introduced into PE successfully, and the average contents of MGF-Ct24 E in PEM was about 1.39 μmol/g.2) The Tg and decomposition temperature of the synthetic polymer PEM were 36.56℃ and 315℃, respectively.⑥ The antibacterial properties and cytotoxicity of PEM were evaluated, separately, and the results are as follows:1) Compared with PE, the MIC and MBC of PEM against E.coli and S.aureus increased, but the corresponding concentration of PEM showed a better bactericidal effect than PE.2) SEM and TEM results showed that, PEM has a similar antibacterial mechanism with PE.3) The introduce of MGF-Ct24 E would not only reduce the cytotoxicity of material, but also played the biological functions of MGF-Ct24 E.⑦ We chosen two kinds of nano-HA to prepare porous HA ceramic scaffold with a certain mechanical strength at first. Then, PE and PEM were compound onto the surface of scaffold, and the antibacterial properties, biocompatibility of the obtained compounds were evaluated subsequently.1) FTIR and SEM analysis showed that, PE and PEM were introduced onto the surface of porous HA ceramic scaffold successfully.2) The obtained compounds could inhibit or kill the bacteria, which were contact with the scaffold directly.3) CCK-8 and BCA analysis showed that, the obtained compounds could promote osteoblasts proliferation and protein secretion, separately.