Rational design of phosphonate/quaternary amine block polymer as an high-efficiency antibacterial coating for metallic substrates

doi:10.1016/j.jmst.2020.05.060

Abstract

Abstract:

Developing advanced technologies to address the bacterial associated infections is an urgent requirement for metallic implants and devices. Here, we report a novel phosphonate/quaternary amine block polymer as the high-efficiency antibacterial coating for metallic substrates. Three pDEMMP-b-pTMAEMA block polymers that bearing identical phosphonate segments (repeat units of 15) but varied cationic segments (repeat units of 8, 45, and 70) were precisely prepared. Stable cationic polymer coatings were constructed on TC4 substrates based on the strong covalent binding between phosphonate group and metallic substrate. Robust relationship between the segment chain length of the polymer coating and the antibacterial property endowed to the substrates have been established based on quantitative and qualitative evaluations. Results showed that the antibacterial rate of the modified TC4 surface were 95.8 % of S. aureus and 92.9 % of E. coli cells attached. Interestingly, unlike the cationic free polymer or cationic hydrogels, the surface anchored cationic polymers do compromise the viability of the attached C2C12 cells but without significant cytotoxicity. In addition, the phosphonate/quaternary amine block polymers can be easily constructed on titanium, stainless steel, and Ni/Cr alloy with significantly improved antibacterial property, indicating the generality of the block polymer for surface antibacterial modification of bio-metals.

Key words: Block polymers, Polymeric coating, Metallic substrates, Polycations, Antibacterial property

Li Liu, Wan Peng, Xiao Zhang, Jiangmei Peng, Pingsheng Liu, Jian Shen. Rational design of phosphonate/quaternary amine block polymer as an high-efficiency antibacterial coating for metallic substrates[J]. J. Mater. Sci. Technol., 2021, 62: 96-106.

Figures/Tables 13

Fig. 1. Schematic illustration of the construction of pDEMMP-b-pTMAEMA copolymers as antibacterial coating on titanium alloy substrates.

Fig. 2. Preparation of pDEMMP-b-pTMAEMA block polymers through a three-step process. (a) Preparation of pDEMMP polymer as the macro-CTA through RAFT polymerization. (b) Preparation of pDEMMP-b-pDMAEMA precursor block polymers through RAFT polymerization. (c) Preparation of cationic polymers (pDEMMP-b-pTMAEMA) through the quaternization of pDEMMP-b-pDMAEMA block copolymers.

Table 1 Synthesis and chemical composition of the block copolymers.

Polymers^a	Feeding mol. ratio n(monomer):n(CTA)^b: n(AIBN)	Theoretic degree of polymerization	Actual degree of polymerization^c	Yield (%)
pDEMMP₁₅	20:1:0.5	20	15	69
pDEMMP₁₅-b-pDMAEMA₈	15:1:0.5	15	8	69
pDEMMP₁₅-b-pDMAEMA₄₅	70:1:0.5	70	45	60
pDEMMP₁₅-b-pDMAEMA₇₀	100:1:0.5	100	70	63

Fig. 3. 1H NMR spectra of pDEMMP-b-pDMAEMA and pDEMMP-b-pTMAEMA block polymers with varied segment lengths. (a) pDEMMP15. (b) pDEMMP15-b-pDMAEMA8. (c) pDEMMP15-b-pDMAEMA45. (d) pDEMMP15-b-pDMAEMA70. (e) pDEMMP15-b-pTMAEMA8 (P8). (f) pDEMMP15-b-pTMAEMA45 (P45). (g) pDEMMP15-b-pTMAEMA70 (P70).

Fig. 4. XPS analysis of TC4 substrates. (a) Survey scan spectra of the TC4 (black), P8 coated block copolymer (red) and the P70 coated block copolymer (blue). (b) N1s, (c) P2p and (d) S2p high resolution scan spectra of TC4 substrates coated with cationic block copolymers. TC4 (black), P8 (red), and P70 (blue).

Table 2 Surface elemental compositions of TC4 substratesa.

	Elemental composition (%)
Sample	Ti	C	O	N	I	P	S
TC4	15.13	31.09	50.88	—	—	—	—
TC4-P ₈	19.67	21.47	54.26	1.55	0.2	1.98	0.87
TC4-P ₇₀	15.85	30.77	48.04	2.11	0.21	2.37	0.65

Fig. 5. (a) Water contact angle of the cationic block copolymers coated TC4 substrates; (b) Images of water contact angle of TC4, TC4-P8, TC4-P45, TC4-P70 substrates. Data are shown as mean ± SEM (n = 6). Statistical significance was determined by one-way ANOVA multiple comparison tests. Pairwise comparisons are statistically significant unless denoted as “ns” (not significant).

Fig. 6. Contact time and cationic segment length dependent antibacterial (S. aureus) property of TC4 substrates. (a) Colony images of S. aureus cells that contacted with TC4 substrates for varied time (2, 4, and 8 h). (b) Colony numbers of S. aureus cells that contacted with TC4 substrates for varied time (2, 4, and 8 h). (c) Antibacterial efficiency of the TC4 substrates coated with different cationic polymers. Data are shown as mean ± SEM (n = 3). Statistical significance was determined by two-way ANOVA multiple comparison tests. Pairwise comparisons are statistically significant as denoted as *.

Fig. 7. Contact time and cationic segment length dependent antibacterial (E. coli) property of TC4 substrates. (a) Colony images of E. coli cells that contacted with TC4 substrates for varied time (2, 4, and 8 h). (b) Colony numbers of E. coli cells that contacted with TC4 substrates for varied time (2, 4, and 8 h). (c) Antibacterial efficiency of the TC4 substrates coated with different cationic polymers. Data are shown as mean ± SEM (n = 3). Statistical significance was determined by two-way ANOVA multiple comparison tests. Pairwise comparisons are statistically significant as denoted as *.

Fig. 8. SEM images of S. aureus and E. coli morphology on various substrates TC4, TC4-P8, TC4-P45, and TC4-P70. Scale bars are 10 μm and 100 nm, respectively. TC4 substrates were immersed in 2 mL of bacterial suspension (106 CFU/mL) and were incubated for 8 h.

Fig. 9. Confocal laser-scanning microscopy images of bacteria (S. aureus and E. coli.) on TC4 and TC4-P70 substrates, respectively. Bacteria cells were seeded on substrates and were incubated for 8 h before staining.

Fig. 10. Cytocompatibilty of the TC4 substrates. (a) Viability of C2C12 cells on different TC4 substrates as determined by CCK-8 kit after 1 and 3 days’ culture. Data are shown as mean ± SEM (n = 3). Statistical significance was determined by two-way ANOVA multiple comparison tests. Pairwise comparisons are statistically significant as denoted as *. (b) Confocal laser-scanning microscopy images of C2C12 cell on TC4 and TC4-P70 after 3 day’s culture.

Fig. 11. Antibacterial properties of pure titanium, stainless steel and Ni/Cr alloys coated with cationic polymers. (a) Colony image, (b) colony number, and (c) antibacterial efficiency of titanium, stainless steel and Ni/Cr alloy substrates against S. aureus. (d) Colony image, (e) colony number, and (f) antibacterial efficiency of titanium, stainless steel and Ni/Cr alloy substrates against E. coli. Data are shown as mean ± SEM (n = 3). Statistical significance was determined by two-way ANOVA multiple comparison tests. Pairwise comparisons are statistically significant as denoted as *.

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