Antibacterial activity and preparation of quaternary ammonium grafted polyphosphazene microspheres
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摘要: 细菌感染引起的疾病问题在世界范围内引起广泛的关注。抗生素虽然能有效治疗细菌感染，但是不合理的使用及滥用会导致细菌产生耐药性。因此，解决细菌耐药性问题并研发出安全高效的非抗生素抗菌剂显得尤为迫切。通过在生物可降解型环交联型聚(环三膦腈-共-聚乙烯亚胺)微球(PHP)表面上接枝环氧丙基十二烷基二甲基氯化铵(DDEAC)，成功制备了环交联型聚(环三膦腈-共-聚乙烯亚胺)接枝季铵盐微球(PHPD)。采用FTIR、XPS、TG、TEM和FESEM对微球的结构与形貌进行了表征分析，并研究了其抗菌活性和细胞毒性。实验结果表明，改性抗菌微球PHPD(50 μg/mL)对大肠杆菌(E.coli)和金黄色葡萄球菌(S.aureus)的抗菌率均达97.3%。复合材料克服了单独使用季铵盐DDEAC材料的高毒性缺陷，并且在实现高效抗菌的同时也具有很好的细胞相容性。因此，本研究对于开发安全高效的纳米抗菌剂具有一定的指导意义。Abstract: Disease problems caused by bacterial infections have resulted in widespread concern worldwide. Although antibiotics can effectively treat bacterial infections, unreasonable use and abuse can cause bacteria to develop drug resistance. Therefore, it is particularly urgent to solve the problem of bacterial drug resistance and develop safe and efficient non-antibiotic antibacterial agents. We grafted epoxypropyl dodecyl dimethyl Ammonium chloride (DDEAC) the surface of biodegradable cyclic cross-linked poly (cyclotriphosphazene-co-polyethyleneimine) microspheres (PHP), and successfully prepared ring-crosslinked poly (cyclotriphosphazene-co-polyethyleneimine) grafted quaternary ammonium salt microspheres (PHPD). In addition, using FTIR, XPS, TG, TEM and FESEM to characterize and analyze the structure and morphology of the microspheres, and studying its antibacterial activity and cytotoxicity. The experimental results show that the modified antibacterial microsphere PHPD (50 μg/mL) has an antibacterial rate of 97.3% against E.coli and S.aureus. The material overcomes the high toxicity defect of using the quaternary ammonium salt DDEAC material alone, and achieves high-efficiency antibacterial while also has good cell compatibility. Therefore, this research has certain guiding significance for the development of safe and efficient nano-antibacterial agents.
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Key words:
- PHP /
- PHPD /
- DDEAC /
- Antibacterial activity /
- cytotoxicity
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图 1 环交联型聚(环三膦腈-共-聚乙烯亚胺)微球(PHP)及交联型聚(环三膦腈-共-聚乙烯亚胺)接枝季铵盐微球(PHPD)的制备过程
Figure 1. Preparation process of biodegradable cyclic cross-linked poly(cyclotriphosphazene-co-polyethyleneimine) microspheres (PHP) and ring-crosslinked poly (cyclotriphosphazene-co-polyethyleneimine) grafted quaternary ammonium salt microspheres (PHPD)
图 2 环氧丙基十二烷基二甲基氯化铵(DDEAC)、PHP及PHPD的FTIR图谱
Figure 2. FTIR spectra of dodecyl dimethyl Ammonium chloride (DDEAC), PHP and PHPD
图 3 PHP (a) 和PHPD (c) 的SEM图像、PHP (b) 和PHPD (d) 的TEM图像
Figure 3. SEM images of PHP (a) and PHPD (c), TEM images of PHP (b) and PHPD (d)
图 4 PHP的XPS全谱 (a) 、N 1s谱( b) 和C 1s谱 (c)； PHPD的XPS全谱 (d) 、N 1s谱 (e) 和C 1s谱 (f)
Figure 4. XPS spectra of PHP of a survey scan (a), N 1s (b) and C 1s (c); PHPD of a survey scan (d), N 1s (e) and C 1s (f)
图 5 PHP、PHPD和DDEAC的TG曲线 (a) 和PHP的DSC曲线 (b)
Figure 5. TG curves (a) of PHP, PHPD, DDEAC and DSC curve (b) of PHP
图 6 空白对照 (a)、PHP处理 (b)、DDEAC处理 (c) 和PHPD处理 (d) 大肠杆菌的SEM图像；空白对照 (e)、PHP处理 (f)、DDEAC处理 (g) 和PHPD处理 (h)大肠杆菌的TEM图像
Figure 6. SEM images of E.coli cells treated by blank (a), PHP (b), DDEAC (c) and PHPD (d); TEM images of E. coli cells treated by blank (e), PHP (f), DDEAC (g) and PHPD (h)
图 8 PHP、PHPD和DDEAC对NIH-3T3细胞的毒性曲线
Figure 8. Cytotoxicity of PHP、PHPD and DDEAC on NIH-3T3 cells
图 7 空白对照 (a)、PHP处理 (b)、DDEAC处理 (c) 和PHPD处理 (d) 金黄色葡萄球菌的SEM图像；空白对照 (e)、PHP处理 (f)、DDEAC处理 (g) 和PHPD处理 (h)金黄色葡萄球菌的TEM图像
Figure 7. SEM images of S.aureus cells treated by blank (a), PHP (b), DDEAC (c) and PHPD (d); TEM images of S. aureus cells treated by blank (e), PHP (f), DDEAC (g) and PHPD (h)
图 9 在50 μg/mL样品浓度下相互作用24 h后NIH-3T3细胞的形态变化
Figure 9. Evolution of morphologies of NIH-3T3 cells after the interaction for 24 h under the concentration of 50 μg/mL
表 1 PHP与DDEAC不同投料质量比的碳氮含量
Table 1. Carbon and nitrogen contents of different feed mass ratios of PHP and DDEAC
PHPD N/wt% C/wt% C/N m(PHP)∶m(DDEAC)=1∶0 24.59 34.56 1.40 m(PHP)∶m(DDEAC)=5∶1 24.25 38.71 1.59 m(PHP)∶m(DDEAC)=3∶1 24.86 40.19 1.61 m(PHP)∶m(DDEAC)=1∶1 22.19 39.22 1.76 m(PHP)∶m(DDEAC)=1∶3 23.13 38.22 1.65 m(PHP)∶m(DDEAC)=1∶5 23.76 35.76 1.50 
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表 2 PHP、DDEAC和PHPD的抗菌活性
Table 2. Antibacterial activity of PHP, DDEAC and PHPD
Strain Sample 5 μg/mL 10 μg/mL 25 μg/mL 50 μg/mL E.coli Control 0 0 0 0 PHP (12±4.2)% (35.6±3.6)% (73.8±0.8)% (93.4±0.2)% DDEAC (50.6±7.5)% (99.0±0.1)% 100% 100% PHPD (84.9±2.9)% (90.9±0.8)% (93.2±1.2)% (97.3±1.1)% S.aureus Control 0 0 0 0 PHP (28.9±3.4)% (40.2±2.8)% (81.9±0.7)% (94.1±1.9)% DDEAC (62.1±0.8)% (99.8±0.3)% 100% 100% PHPD (86.8±1.3)% (88.1±1.9)% (93.8±0.9)% (97.3±0.3)%
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表 3 大肠杆菌细胞外液的K+、Ca2+和Mg2+的浓度
Table 3. Concentration of K+, Ca2+ and Mg2+ in the treated cell extracellular fluid
Sample Time/h K+/(mg·L−1) Ca2+/(mg·L−1) Mg2+/(mg·L−1) Control 0 0.0206 0.0644 0.0050 2 0.0285 0.0757 0.0039 4 0.0349 0.1123 0.0065 6 0.0312 0.0989 0.0088 PHPD 0 0.0336 0.0887 0.0076 2 0.0362 0.1009 0.0106 4 0.0512 0.1258 0.0102 6 0.0653 0.1234 0.0138
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