High Throughput Screening of Glycopolymers: Balance between

ACS Macro Lett. , 2019, 8, pp 326–330. DOI: 10.1021/acsmacrolett.9b00091. Publication Date (Web): March 8, 2019. Copyright © 2019 American Chemical...
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High Throughput Screening of Glycopolymers: Balance between Cytotoxicity and Antibacterial Property Yuqing Zheng,† Yan Luo,‡ Kai Feng,† Weidong Zhang,*,† and Gaojian Chen*,†,‡ †

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Center for Soft Condensed Matter Physics and Interdisciplinary Research and School of Physical Science and Technology, Soochow University, Suzhou 215006, People’s Republic of China ‡ State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, People’s Republic of China S Supporting Information *

ABSTRACT: To search for synthetic agents with low cytotoxicity and good antibacterial activity is essential for antimicrobial applications. Here we report a high throughput technique that carried out in multiwell plates via recyclable-catalyst-aided, opened-to-air, and sunlight-photolyzed RAFT (ROS-RAFT) polymerization. By using this method, three key monomers (MAG the sugar unit, DMAPMA the positively charged monomer, and DEMAA the hydrophobic monomer) can be polymerized in a controlled manner to afford glycopolymers. This simple high throughput technology is used to synthesize glycopolymers with variable compositions. The bacterial adhesion/killing ability and cytotoxicity of synthesized polymers have been evaluated, and glycopolymers with certain composition can achieve a balance of low cytotoxic and good antibacterial activity.

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bacterial membrane cell surface; for example, several types of fimbriae exist on E. coli, such as type 1, type P, type S, and type G, each having specific affinity toward different sugar units.18 Carbohydrate can bring high biocompatibility, better adhesion ability, and selective properties.19−24 Therefore, it is an ideal choice to introduce carbohydrate unit as hydrophilic component into the peptide-mimetic antimicrobial polymer. Based on the above, we propose an idea to screen optimum and cell-friendly antibacterial glycopolymers bearing cationic and hydrophobic segments that are able to achieve a balance between germicidal properties and low cytotoxicity by changing the ratio of these three different components. High throughput (HTP) as a time-saving and convenient technique has been widely applied in the synthesis and screening of a number of new functional polymeric materials in recent years. Many excellent works in high throughput synthetic preparation polymers have been reported.25−43 Among them, the open-to-air polymerization facilitates the screening process extensively. For example, the group of Boyer and Chapman have recently shown the use of PET-RAFT polymerization strategy to prepare high throughput CLRP libraries in the presence of air.37,43 We recently reported a recyclable-catalyst-aided, opened-to-air, and sunlight-photolyzed RAFT (ROS-RAFT)44 polymerization that we believed is suitable for the HTP in an open reactor, such as multiwell plates and beneficial for the preparation of biomacromolecules.

acteria play an important role in nature, which have a significant influence on the living environment. Apart from the positive part bacteria participate in, that is, digesting nutrients, producing growth factors for animals and plants, manufacturing of fermented foods, acting as a medical source (some antibiotics and vaccines), and so on, bacteria are also threatening pathogens to the living body. Therefore, a rational antibacterial strategy is increasingly required. Use of antibiotics is one of the effective solutions. However, the misuse of antibiotics, the emergence of antibiotic-resistant bacteria “superbugs”, and the slow development of new antibiotics are becoming urgent issues for public health and environment.1−4 In the search for new antimicrobial agents, antimicrobial peptide (AMP) is emerging as a promising alternate for antibiotics, which has a broad antibacterial spectrum and is barely antibiotic-resistant. Almost all AMPs have amphiphilic and cationic moieties in their structure, which endow AMPs key properties that lead to effective antibacterial performance such as aqueous solubility (hydrophilic part), passing/disrupting cell membranes (hydrophobic and positive part) resulting in the death of bacteria.5 However, the pervasive application of antibacterial peptides as antibacterial agents is hampered by their high manufacturing cost and low stability due to proteolytic degradation and cytotoxicity. As a consequence, research on the preparation of synthetic antimicrobial peptides and new molecules mimicking natural antimicrobial peptides have gradually emerged, and polymers inspired by natural antibacterial peptides are one of the major types.6−17 It is reported that there are many carbohydrate receptors that exist on the © XXXX American Chemical Society

Received: January 31, 2019 Accepted: March 7, 2019

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DOI: 10.1021/acsmacrolett.9b00091 ACS Macro Lett. 2019, 8, 326−330

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ACS Macro Letters Scheme 1. Illustration of the HTP Screening of Antibacterial Cell-Friendly Glycopolymers

A large number of polymers can be prepared at one time, which is of great practicability to control different proportions for the synthesis and screening of polymers. Herein for the screening of glycopolymers with a balance between low cytotoxicity and optimal antibacterial ability, we have developed a new HTP technique utilizing ROS-RAFT polymerizations. In this regard, we conducted polymerization experiments via the open-to-air light-induced RAFT method. We applied this method to the copolymerization of three different monomers in the 96-well plate to prepare a series of glycopolymers with different predetermined ratios. Glucose/glucosamine-based monomer is hydrophilic and can specifically bind to a variety of protein receptors on some cell surfaces, including the surface of Escherichia coli. Therefore, we chose 2-(methacrylamido) glucopyranose (MAG) as the monomer providing the sugar unit to the polymer. Besides the sugar monomer MAG, we selected N-[3-(dimethylamino)propyl] methacrylamide (DMAPMA) as the positively charged monomer and N,Ndiethylmethacrylamide (DEMAA) monomer to offer hydrophobic properties in the current research. A range of glycopolymers were synthesized in the 96-well plate (volume = 1.1 mL), under the simulating sunlight (light intensity = 11 mw/cm2), and at 25 °C in a dry bath thermostat (Scheme 1, Figure S1). Three groups of sugar-containing polymers were synthesized by changing the proportion of different monomers with the same target chain length (DP = 200). Kinetics of the polymerization was first investigated. Taking the polymerization [MAG]0:[DMAPMA]0:[DEMAA]0 = 100:90:10 ([MAG]0 = 0.415 mol/L) as an example. Crude samples of the polymerization mixture were taken for 1 H NMR measurement (Figure 1B). The linear plot of Ln([M]0/[M]) versus time show that there exists a short induction time,44 and the period is shorter than our previous report due to stronger light intensity. We further calculated the conversions of three monomers during the polymerization (Table S3) and found that for MAG is slightly slower due to its relative bulky property,45 but the difference is small. The molecular weight distributions of the polymers obtained by aqueous SEC (size exclusion chromatography) were relatively low (Figures 1C and S2). The obtained molecular weights from SEC are much lower than the theoretical values, due to the difference between sample and standards, especially the unique folded conformation of the glycopolymers.46 Typical peaks from the three monomers can be found in NMR (Figure

Figure 1. (A) 1H NMR spectrum (D2O) of typical glycopolymer; (B) Ln([M]0/[M]) of typical polymerization monitored by 1H NMR; (C) Dependence of the molecular weight (Mn) and molecular weight distribution (Mw/Mn) on total monomer conversions (by gravimetry) for the polymerization. Polymerization conditions: [MAG]0:[DMAPMA]0:[DEMAA]0 = 100:90:10, [MAG]0 = 0.415 mol/L, in the presence of air, 25 °C.

1A), and their ratios are calculated to be consistent with feeding ratios (Table S2). After obtaining the purified polymers, we tested the bacterial adhesion and antibacterial ability of the glycopolymers. The adhesion ability of the polymers to bacteria was evaluated by turbidimetry experiments. The ability of polymers to bind to E. coli was extrapolated from their ability to form bacterial aggregates. And the tests were carried out for the mixture of polymer solution (1 mg/mL) and Escherichia coli sterile aqueous suspension (OD = 0.4, 600 nm) in a 96-well plate.49 The adhesion ability of these synthesized polymers to bacteria is shown in Figure 2A. ODmax represents the maximum optical density (OD) value obtained by micro reader in the process of mixing the polymer solutions and suspension of E. coli. We can see that, keeping the chain length unchanged, with the decrease of sugar components in polymer, the cationic and hydrophobic components increase and its bacterial adhesion capacity show a trend of increase, indicating that the positively 327

DOI: 10.1021/acsmacrolett.9b00091 ACS Macro Lett. 2019, 8, 326−330

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ACS Macro Letters

85%) to test the cytotoxicity of the antibacterial process. The experiment was carried out by CCK-8 method, using L929 as the model cell. The result is shown in Figure 3A. The area of

Figure 3. (A) Antibacterial performance on E. coli and cytotoxicity of selected glycopolymers; (B) Typical agar plates of E. coli and S. aureus treated with selected polymers for 3 h.

Figure 2. (A) Adhesion ability and (B) antibacterial ability of glycopolymers. [MAG]0:[DMAPMA]0:[DEMAA]0 = 100:X:100 − X (X = 10, 20, 30, 40, 50, 60, 70, 80, 90), [MAG]0:[DMAPMA]0: [DEMAA]0 = 67:X:133 − X (X = 13, 27, 40, 53, 67, 80, 93, 106, 120), [MAG]0:[DMAPMA]0:[DEMAA]0 = 18:X:182 − X (X = 18, 36, 55, 73, 91, 109, 127, 145, 164).

the red color box are two polymers, samples 1-2 ([MAG]0: [DMAPMA]0:[DEMAA]0 = 100:20:80) and 1-3 ([MAG]0: [DMAPMA]0:[DEMAA]0 = 100:30:70), that have good bactericidal properties, and their cell viability is as high as 98% and 87%, which we believe are two good examples that can achieve a balance of low cytotoxic and good antibacterial activity. Furthermore, we tried these selected polymers on Gram-positive bacterial S. aureus, and the results are shown in Figure 3B; polymers with a higher content of positively charged DMAPMA (1-7, 1-8, 1-9) show notable antibacterial ability toward both E. coli and S. aureus, while those containing more MAG (1-2, 1-3, 1-4) show obvious ability to kill E. coli rather than S. aureus. This selectivity is possible due to the fimH protein exist on E. coli that possesses carbohydrate recognition sites (CRS) with high affinity for glucose,50 and the protein is absent on S. aureus. In conclusion, by using the open-to-air HTP method, a series of polymers can be conveniently synthesized, and we find out polymers with more sugar and hydrophobic components, even if only containing a relatively low proper proportion of positively charged monomers, can also have good bactericidal ability toward certain bacteria and low cytotoxicity. Preliminary results show the potential for screening polymers with selective antibacterial properties to identify probiotics and pathogens, which will be carried out in our future work. We also believe this convenient technique can be applied in the screening of many other polymers with optimal functionality.

charged and hydrophobic components have a greater impact on the adhesion properties of polymers and bacteria.47 Next, we tested the antibacterial ability of these synthetic polymers, and the difference of antibacterial performance of these polymer solution was conducted by using colony counting assay. Through the analysis of experimental results, we found that sterilization ability is not just related to the adhesion ability. As shown in Figure 2B, the areas of the blue and red color boxes are the range of polymers which have better antibacterial performance demonstrating low survivable rate of bacteria. For the group of polymers with least sugar components and most cationic and hydrophobic parts (blue box shown in Figure 2A,B), the sterilization ability is positively related to the adhesion ability. In the meantime, although containing lower proportion of positively charged monomers, and having moderate bacterial adhesion ability (red box shown in Figure 2A, Table S4) polymers containing most saccharide components still have good bactericidal ability (red box shown in Figure 2B and Table S5). From the perspective of biological applications, although the positively charged components will bring better bactericidal performance, it is not always purely positive to the biological sterilization process, as it may lead to an increase in cytotoxicity.48 The addition of sugar-containing monomer MAG brings the advantages of low toxicity toward the sterilization process. We selected 12 polymers of different monomer ratios with notable antibacterial performance (sterilization rate above 328

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.9b00091. Details of the experimental section, equipment, SEC of polymers, adhesion and antibacterial assay, and cytotoxic assay (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Weidong Zhang: 0000-0002-6837-3060 Gaojian Chen: 0000-0002-5877-3159 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the National Natural Science Foundation of China (Grants 21774084), the Natural Science Foundation of Jiangsu Province (Grants BK20161208 and BK20171208), and the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions for financial support.

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