Dual functional β-peptide polymer-modified resin beads for bacterial killing and endotoxin adsorption
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Bacterial infections and endotoxin contaminations are serious problems in the production/manufacture of food, water, drinks, and injections. The development of effective materials to kill bacteria and adsorb endotoxins, particularly those caused by gram-negative bacteria, represents a major step toward improved safety. As synthetic mimic of host defense peptides, β-peptide polymers are not susceptible to bacterial resistance and exhibit potent bacteria-killing abilities upon antibiotic-resistant bacteria. This study investigated the potential of synthetic β-peptide polymer-modified polyacrylate (PA) beads to kill bacteria and remove endotoxin, i.e. lipopolysaccharide (LPS), produced by these bacteria.
Synthetic β-peptide polymer-modified PA beads displayed strong antimicrobial activity against Escherichia coli and methicillin-resistant Staphylococcus aureus, as well as excellent biocompatibility. In addition, these β-peptide polymer-modified beads removed around 90% of the endotoxins, even at 200 EU/mL of LPS, a very high concentration of LPS.
β-peptide polymer-modified PA beads are efficient in bacterial killing and endotoxin adsorption. Hence, these modified beads demonstrate the potential application in the production/manufacture of food, water, drinks, and injections.
methicillin-resistant Staphylococcus aureus
host defense peptides
colony forming unit
scanning electron microscope
Bacterial contamination of food packages, water treatment membranes, industrial pipes, and drug injection and medical devices is a serious problem globally and poses a threat to their biosafety and effectiveness [1, 2, 3, 4, 5]. To reduce or prevent bacterial contamination, antimicrobial drugs and antimicrobial coatings are widely used [6, 7, 8, 9]. Unfortunately, indiscriminate use of antimicrobials has led to the emergence and spread of drug-resistant bacteria, which poses a challenge to human health [10, 11, 12, 13]. In addition, biosafety-related factors such as immunomodulation are also very important.
Endotoxins, lipopolysaccharide (LPS) that function as major pathogenic immune factor, are released from the outer cell membrane of Gram-negative bacteria in response to an attack by antimicrobial agents. Endotoxins can activate complex immune effectors to generate a hyperinflammatory response and even provoke severe endotoxic shock and multiorgan dysfunction [14, 15, 16, 17, 18]. Therefore, multifunctional antibacterial materials are highly desirable for both efficient bacterial killing and biosafety considerations [19, 20, 21, 22, 23, 24, 25].
In contrast to conventional antibiotics, host-defense peptides (HDPs) have low susceptibility to antimicrobial resistance. Given this advantage, HDPs have received much research attention [26, 27, 28, 29, 30]. The versatile biological functions, such as antimicrobial activity combined with anti-inflammatory properties, of HDPs have made them promising candidates in relieving acute inflammation via inactivating or neutralizing endotoxins, in addition to killing bacteria [31, 32, 33, 34]. The amphipathic structure of HDPs plays an important role in the process of endotoxin removal as well as in bacterial killing through hydrophobic and electrostatic interaction with toxic lipid A. This interaction occurs when the positively charged fragments within HDPs attract negatively charged phosphates of lipids A, and the hydrophobic fragments of HDPs bind with lipid A fatty acid moieties. However, HDPs derived from diverse sources have similar shortcomings: low stability upon proteolysis and a high cost. To address these problems, a series of synthetic mimics of HDPs have been developed. Several studies showed that these synthetic mimics of HDPs exhibited high endotoxin neutralization and killing efficacy against bacteria, thereby showing strong potential in antibacterial applications [35, 36, 37].
As synthetic mimics of HDPs, amphipathic β-peptide polymers display broad-spectrum and potent antimicrobial activities, in addition to favorable solution [38, 39, 40, 41, 42] and surface biocompatibility [43, 44]. In previous study, a thiol-terminated β-peptide polymer (50:50 DM-CH) was successfully modified to the flat surfaces of gold  and variable biomedical materials  and displayed excellent antimicrobial activity. In this study, we modified 50:50 DM-CH to the spherical surface of amino-functionalized polyacrylate (PA) resin beads and demonstrated their function in efficient bacterial killing and endotoxin adsorption.
Results and discussion
Encouraged by these results, we investigated the antimicrobial ability of polymer-modified beads against E. coli and MRSA in the presence of serum, using 50% fetal bovine serum (FBS) in the assay medium. We observed 99.9% bacterial killing of both E. coli and MRSA by ≥ 50 mg beads per sample were used (Fig. 2b). We used scanning electron microscope (SEM) to assess morphological changes of E. coli and MRSA, incubated with the β-peptide polymer modified PA resin beads for 2.5 h. As compared to the intact membrane of bacteria incubated with bare beads, conspicuous shrinkage and damage of the bacterial membrane were observed among bacteria incubated with the β-peptide polymer-modified beads (Fig. 2c). This observation appointed to a membrane-active antimicrobial mechanism similar to that observed in our previous studies on the antimicrobial abilities of gold and polyurethane surfaces coated with β-peptide polymers [43, 44].
We successfully modified PA beads with a synthetic β-peptide polymer, thiol-terminated 50:50 DM-CH. The resulting resin beads exhibited potent antibacterial activity against both Gram-negative E. coli and Gram-positive MRSA. Additionally, the modified beads demonstrated the ability for endotoxin adsorption. The biocompatibility and ease of synthesis of these polymer-modified beads point to their potential application as dual-functional materials for antibacterial and endotoxin adsorption.
Materials and methods
PA resin beads were purchased from Tianjin Nankai HECHENG S&T Co.,Ltd; bromoform, chlorosulfonyl isocyanate, trifluoroacetic anhydride, di-tert-butyl pyrocarbonate were purchased from Adamas-beta; Triphenylmethyl chloride and N-hydroxy succinimide (NHS) were obtained from Meryer Technologies in China; PBS was purchased from Thermo Fisher Scientific; LPS from Escherichia coli O111:B4, FITC-conjugates was purchased from Sigma-Aldrich; all others reagents and solvents were purchased from General-Reagent. In this study, two types of bacteria were used for in vitro antimicrobial test including Escherichia coli (E. coli ATCC 25922) and Staphylococcus aureus (S. aureus USA 300, methicillin-resistant strain, MRSA); NIH-3T3 fibroblast cells (3T3 ATCC CRL-1658) were obtained from the Cell Bank of Typical Culture Collection of Chinese Academy of Sciences (Shanghai, China) and were used for cytotoxicity study. Synthesized chemicals were purified using a SepaBean machine equipped with Sepaflash columns produced by Santai Technologies Inc. in China. CDCl3 or D2O were used as the solvent to collect the 1H NMR spectra on a Bruker spectrometer at 400 MHz. 1H NMR chemical shifts were referenced to the resonance for TMS internal standard for CDCl3 and residual protonated solvent for D2O; The mass spectrum data of compounds were collected using an Agilent HPLC 1100/MS G1956B mass spectrometer. Element analysis of the β-peptide polymer-modified PA resin beads was acquired using Thermo Fisher ESCALAB 250XI X-ray photoelectron spectroscopy (XPS). Morphology of bacteria on the modified resin beads was observed on a Hitachi S-4800 Field Emission Scanning Electron Microscope (FESEM). The TAL assay was provided by Xiamen Bioendo Technology. Co., Ltd. (Xiamen, China).
Synthesis of β-lactam monomers and poly-β-peptides
β-lactam monomers and poly-β-peptides were prepared by following the procedure in the literature [43, 45, 46]. The details are given in the Additional file 1, Synthesis S1. Synthesis of racemic β-lactam monomer (±) DMβ; Synthesis S2. Synthesis of β-Lactam monomers (±)-CHβ; Synthesis S3. Synthesis of polymerization co-Initiator; Synthesis S4. Synthesis of β-peptide polymers; Figure S1. 1H NMR spectrum of monomer (±) DMβ; Figure S2. 1H NMR spectrum of monomer (±) CHβ; Figure S3. 1H NMR spectrum of co-initiator; Figure S4. 1H NMR spectrum of β-peptide polymer 50:50 DM-CH.
Synthesis of the surface linker
3-Maleimidopropionic acid N-hydroxysuccinimide ester (MalOSu) was prepared according to the literature . The details are given in the Additional file 1, Synthesis S4. Synthesis of the surface linker; Figure S5. 1H NMR spectrum of surface linker MalOSu.
Synthesis and characterization of poly-β-peptide immobilized on the surface of PA resin beads
Examination on bactericidal efficacy of polymer modified surface in PBS and serum
SEM characterization of bacterial morphology
Bacterial cell suspension at the end of the above antimicrobial assay was collected and was fixed with 4% glutaraldehyde in phosphate buffer (PB) at 4 °C overnight. Then the fixed cells were rinsed with PBS three times and were dehydrated using a graded ethanol series of (30–100% ethanol). The sample was dried under N2 and was used directly for FESEM characterization.
FITC-LPS binding assay on polymer modified surface
Adsorption of endotoxin (LPS) in serum
20 mg of polymer-modified beads were incubated in 50% FBS with 0–200 EU/mL endotoxin at 100 rpm for 3 h. The Chromogenic Tachypleus Amebocyte Lysate kit (Xiamen Bioendo Technology company, China) was used to measure endotoxin. Samples were heated at 70 °C to precipitate proteins followed by testing endotoxin concentration according to the manufacture’s introduction (the raw data of figures in Additional file 3).
Hemolysis assay on polymer modified surface
Statistic analysis of the data was conducted using ANOVA and Tukey’s HSD posthoc test. A p value ≤ 0.05 is considered as statistically significant.
RHL and FD proposed the research. YXQ, YS, RHL, and FD designed the experiments. YXQ, DS, QF, LZY carried out the preparation of monomer and polymer, synthesis of modified beads, XPS analysis, antimicrobial assay, hemolysis assay and then interpreted the data and drafted the manuscript. YS and TYL did antimicrobial assay in serum and TAL assay. LQL conducted the synthesis of the surface linker. NS performed cytotoxicity test, JYX made SEM characterization. All authors read and approved the final manuscript.
This research was supported by the National Natural Science Foundation of China (No. 21574038, 21861162010), the National Key Research and Development Program of China (2016YFC1100401), the Natural Science Foundation of Shanghai (18ZR1410300), the “Eastern Scholar Professorship” from Shanghai local government (TP2014034), the national special fund for State Key Laboratory of Bioreactor Engineering, the Fundamental Research Funds for the Central Universities (22221818014). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information files.
The authors declare that they have no competing interests.
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