Effect of radiation sterilisation on the structure and antibacterial properties of antimicrobial peptides

doi:10.12336/biomatertransl.2023.01.007

Biomaterials Translational ›› 2023, Vol. 4 ›› Issue (1): 51-61.doi: 10.12336/biomatertransl.2023.01.007

• RESEARCH ARTICLE • Previous Articles

Effect of radiation sterilisation on the structure and antibacterial properties of antimicrobial peptides

Xiaodan Wang¹^,²^,^*(), Qinmei Li¹, Huawei Yang²^,³^,^*()

1 Hygea Medical Technology Co., Ltd., Beijing, China
2 State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin Province, China
3 Department of Applied Chemistry and Engineering, University of Science and Technology of China, Hefei, Anhui Province, China.

Received:2023-01-05 Revised:2023-02-17 Accepted:2023-03-09 Online:2023-03-29 Published:2023-03-28
Contact: * Xiaodan Wang,wxd@hygeamed.com;Huawei Yang,yanghw@ciac.ac.cn.
About author:Xiaodan Wang,wxd@hygeamed.com;
Huawei Yang,yanghw@ciac.ac.cn.

Abstract

Abstract:

Antimicrobial peptides (AMPs) have recently been exploited to fabricate anti–infective medical devices due to their biocompatibility and ability to combat multidrug–resistant bacteria. Modern medical devices should be thoroughly sterilised before use to avoid cross–infection and disease transmission, consequently it is essential to evaluate whether AMPs withstand the sterilisation process or not. In this study, the effect of radiation sterilisation on the structure and properties of AMPs was explored. Fourteen AMPs formed from different monomers with different topologies were synthesised by ring–opening polymerisation of N–carboxyanhydrides. The results of solubility testing showed that the star–shaped AMPs changed from water–soluble to water–insoluble after irradiation, while the solubility of linear AMPs remained unchanged. Matrix–assisted laser desorption/ionisation time of flight mass spectrometry showed that the molecular weight of the linear AMPs underwent minimal changes after irradiation. The results of minimum inhibitory concentration assay also illustrated that radiation sterilisation had little effect on the antibacterial properties of the linear AMPs. Therefore, radiation sterilisation may be a feasible method for the sterilisation of AMPs, which have promising commercial applications in medical devices.

Key words: antibacterial activity, antimicrobial peptides, radiation sterilisation

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Figures/Tables 13

Figure 1. The molecular structure and abbreviation of each AMP in this study. AMP: antimicrobial peptide.

Table 1. Detailed molecular parameters of the antimicrobial peptides

Sample	Arm No.	Designed Lys content (%)	Actual Lys content^a (%)	Mn designed molecular weight (Da)	Mn determined molecular weight (Da)^b	? determined molecular weight^b
LP₁	1	70	70.8	6219.9	6200	1.07
LP₂	1	60	61.4	5976.6	6000	1.14
LP₃	1	50	51.2	5733.2	5700	1.24
G₂–P₁	16	70	71	101155.6	111200	1.05
G₂–P₂	16	60	60	97262.3	97300	1.08
G₂–P₃	16	50	48.2	93369	93400	1.12
LV₁	1	70	68.6	5787.5	5800	1.11
LV₂	1	60	59.3	5400	5500	1.09
LV₃	1	50	49	5012.5	5000	1.18
G₂–V₁	16	70	72.3	94236.4	94300	1.02
G₂–V₂	16	60	59.1	88036.7	88000	1.07
G₂–V₃	16	50	50	81837	81900	1.14
LL₃₀	1	100	100	6949.9	6900	1.05
G₂–L₃₀	16	100	100	112835.4	113000	1.13

Table 2. Solubility of the antimicrobial peptides before and after radiation sterilisation

Sample	Before radiation sterilisation	After radiation sterilisation
Sample	PBS buffer	PBS buffer	PBS/AcOH	HFP
LP₁	+	+	+	+
LP₂	+	+	+	+
LP₃	+	+	+	+
G₂–P₁	+	–	±	+
G₂–P₂	+	–	–	+
G₂–P₃	+	–	–	+
LV₁	+	+	+	+
LV₂	+	+	+	+
LV₃	+	+	+	+
G₂–V₁	+	–	–	±
G₂–V₂	+	–	–	±
G₂–V₃	+	–	–	±
LL₃₀	+	+	+	+
G₂–L₃₀	+	–	±	+

Figure 2. Molecular structure and molecular weight information of all linear AMPs. AMP: antimicrobial peptide.

Figure 3. The spectra of LL30 before (A) and after (B) irradiation under matrix–assisted laser desorption/ionisation time of flight (MALDI–TOF) mass spectrometry.

Figure 4. The spectra of LP1–LP3 before (A1–C1) and after (A2–C2) irradiation under matrix–assisted laser desorption/ionisation time of flight (MALDI–TOF) mass spectrometry.

Figure 5. The spectra of LV1–LV3 before (A1–C1) and after (A2–C2) irradiation under matrix–assisted laser desorption/ionisation time of flight (MALDI–TOF) mass spectrometry.

Figure 6. The minimum inhibitory concentrations of LL30 before and after irradiation. (A) Staphylococcus aureus (S. aureus). (B) Escherichia coli (E. coli). OD: optical density.

Figure 7. The minimum inhibitory concentrations of LP1–LP3 (A–C) before and after irradiation. E. coli: Escherichia coli; OD: optical density; S. aureus: Staphylococcus aureus.

Figure 8. The minimum inhibitory concentrations of LV1–LV3 (A–C) before and after irradiation. E. coli: Escherichia coli; OD: optical density; S. aureus: Staphylococcus aureus.

Table 3. The minimum inhibitory concentrations (μg/mL) of antimicrobial peptides against S. aureus and E. coli before and after irradiation

	S. aureus	E. coli
LL₃₀
Before irradiation	16	16
After irradiation	16	16
LP₂
Before irradiation	16	32
After irradiation	16	32
LP₂
Before irradiation	16	32
After irradiation	16	32
LP₃
Before irradiation	16	32
After irradiation	16	32
LV₁
Before irradiation	16	8
After irradiation	16	8
LV₂
Before irradiation	8	16
After irradiation	16	16
LV₃
Before irradiation	16	32
After irradiation	32	64

Figure 1. 1H nuclear magnetic resonance spectroscopy of Nε–tert–butyloxycarbonyl–L–lysine N–carboxyanhydride (Boc–L–Lys–NCA) (A), D–phenylalanine N–carboxyanhydride (D–Phe–NCA) (B) and D, L–valine N–carboxyanhydride (D, L–Val–NCA) (C).

Figure 2. 1H nuclear magnetic resonance spectroscopy of LL30 (A), LP1 (B), LP2 (C), LP3 (D), LV1 (E), LV2 (F), LV3 (G), G2–L30 (H), G2–P1 (I), G2–P2 (J), G2–P3 (K), G2–L30 (L), G2–L30 (M) and G2–L30 (N).

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