Letter pubs.acs.org/macroletters
Polypseudorotaxane Constructed from Cationic Polymer with Cucurbit[7]uril for Controlled Antibacterial Activity Zehuan Huang,† Hongyi Zhang,§,† Haotian Bai,‡ Yunhao Bai,† Shu Wang,‡ and Xi Zhang*,† †
Key Lab of Organic Optoelectronics & Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084, P.R. China ‡ Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P.R. China § Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907-2084, United States S Supporting Information *
ABSTRACT: This letter is aimed to develop a general strategy to fabricate polypseudorotaxanes with controlled antibacterial activity based on cationic polymers. As a proof of concept, the commercially available antibacterial cationic polymer, ε-poly-L-lysine hydrochloride, was chosen for the demonstration. Using host−guest chemistry, cucurbit[7]uril (CB[7]), a water-soluble macrocyclic host, was employed to bind with the positive charge and hydrophobic component on εpoly-L-lysine hydrochlorides for antibacterial regulation. In this way, by tuning the ratio of CB[7] to the cationic polymer, the antibacterial polypseudorotaxane can be obtained, and the antibacterial efficiency can be well tuned from 5% to 100%. This line of research will enrich the field of cationic polymers and polypseudorotaxanes with important functions on precise control over antibacterial activity.
Polyrotaxanes and polypseudorotaxanes, constructed by encapsulation of covalent polymers with macrocyclic hosts, have attracted considerable interest in the fields of supramolecular chemistry, polymer chemistry, and material science because of their unique structures and properties.1,2 In addition to supramolecular architectures of polyrotaxanes and polypseudorotaxanes with diversified approaches, many research works have been focused on bridging the gap between architectures and functions for drug delivery,3,4 conducting materials,5,6 artificial molecular machines,7 gene delivery,8,9 supra-amphiphiles,10,11 and other functional supramolecular systems.12−15 There remain great opportunities to employ polyrotaxanes or polypseudorotaxanes in antibacterial or antifungal therapy. Avoiding accumulation of active antibacterial agents in the environment will greatly decrease the emergence of bacterial resistance from a long-term point of view.16−18 Recently, antibacterial regulation of antibacterial agents to avoid this problem has drawn increasing attention.19−21 Among antibacterial agents, antibacterial cationic polymers, fabricated by positively charged monomers, have been studied for many years in antibacterial treatment.22−33 How to realize control over the antibacterial activity of antibacterial cationic polymers is a big challenge to face. Herein, we have developed a general strategy to fabricate polypseudorotaxanes with controlled antibacterial activity based on cationic polymers (Scheme 1). The positive charges and hydrophobic components on cationic polymers are two key points for these polymers, which can help to bind with the © XXXX American Chemical Society
bacterial surface and destroy the osmotic equilibrium of bacterial cells and thus kill them. Based on host−guest chemistry, many water-soluble macrocyclic hosts can be utilized to bind with the positive charges and hydrophobic components of cationic polymers, thus fabricating polypseudorotaxanes. By tuning the ratio of the macrocyclic host to the cationic polymers, the antibacterial activity may be controlled. To this end, as a proof of concept, the commercially available antibacterial cationic polymer, ε-poly-L-lysine hydrochloride, was selected as the model antibacterial agent. ε-Poly-L-lysine hydrochloride is a natural polymer which is first isolated from culture filtrates of Streptomyces albulus.34−36 This polymer has been widely used as a food preservative in Japan and Korea since the 1980s, and it was approved as a nutritional preservative by the Food and Drug Administration (FDA) of U.S.A. in 2003. As for the macrocyclic host, cucurbit[7]uril (CB[7]) was chosen as the model. Cucurbit[n]urils (CB[n]s) are a family of water-soluble macrocyclic hosts, which have a hydrophobic cavity capable of the binding of one or two guest molecules depending on the size of different CB[n]s.37−42 Among them, CB[7] has good solubility in water and strong affinity with one guest molecule.43−51 Based on host−guest complexations, we employed CB[7] to bind with the positive charge and hydrophobic component on ε-poly-L-lysine hydroReceived: July 25, 2016 Accepted: September 12, 2016
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DOI: 10.1021/acsmacrolett.6b00568 ACS Macro Lett. 2016, 5, 1109−1113
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ACS Macro Letters
Scheme 1. Schematic Presentation of a Strategy to Fabricate Polypseudorotaxanes with Controlled Antibacterial Activity Based on Cationic Polymers and Host−Guest Chemistry
Figure 1. (a) ITC curves and fitted data of the host−guest complexation between CB[7] and L3 in the PBS buffer of pH 6.0 at 25.0 °C. (b) CB[7]dependent 1H NMR titration spectra of the complexation between CB[7] and L3 in PBS D2O buffer of pH 6.0 at 25.0 °C ([L3] = 5.0 mM in all cases).
In order to further confirm the stoichiometry of the host− guest complexation, CB[7]-dependent 1H NMR titration experiments were rationally performed. The solution of 5.0 mM L3 was prepared in PBS buffer at first, and then the white solid of CB[7] was added into this solution in different ratios. After ultrasonication for several minutes, the complexation between L3 and CB[7] could reach the equilibrium. A previous study has investigated the host−guest complexation between lysine itself and CB[7].52 Based on their work, the peaks of protons on L3 after complexation with CB[7] can be assigned. As shown in Figure 1b, all five kinds of protons on the L3 (Figure 1b bottom) were well resolved at the very beginning. After adding CB[7], the intensity of the peaks related to protons 1∼5 significantly decreased, and all of them shifted to the right clearly which indicated that the lysine unit could be partially incorporated into CB[7]’s cavity. However, a series of peaks did not exhibit clear changes in their chemical shifts, indicating that there was at least one or even two lysine units in the polymer chain without complexation in this case. In addition, when the ratio of CB[7] to L3 exceeded 1.0 equiv, the peaks of protons on the L3 did not change. Therefore, the stoichiometric number of the complexation between CB[7] and L3 can be corroborated to be 1.0.
chlorides to construct the polypseudorotaxane for antibacterial regulation. In this way, by tuning the ratio of CB[7] to the cationic polymer, the antibacterial polypseudorotaxane could be obtained, and the antibacterial efficiency could be fine-tuned. We wondered if the host−guest complexation between εpoly-L-lysine hydrochlorides and CB[7] could be formed. To answer this question, isothermal titration calorimetry (ITC) was employed to obtain the thermodynamic information about their complexation. For studying the stoichiometry, ε-poly-Llysine hydrochlorides were characterized by 1H NMR (Figure S1) at first. By end group analysis, the polymerization degree was calculated to be 30, and the molecular weight was 4.96 × 103 g/mol. Besides, every third lysine in the ε-poly-L-lysine hydrochlorides was regarded as one binding motif, and ε-polyL-lysine hydrochloride was briefed as L3 in the following paragraphs. Then, by titrating CB[7] into ε-poly-L-lysine hydrochlorides, we observed an abrupt transition at the ratio of 1.0 equiv of CB[7] to 1.0 equiv of L3 (Figure 1a). Fitting this curve with one set of site binding model, the binding constant of L3 complexed with CB[7] could be calculated to be (3.06 ± 0.16) × 105 M−1. The high binding affinity between ε-poly-Llysine hydrochlorides and CB[7] can guarantee their host− guest complexation in a quantitative manner, leading to the formation of the polypseudorotaxane. 1110
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ACS Macro Letters
10−11 m2/s, respectively. This means the size of L3-CB[7] is only a little bit larger than the size of L3 itself, which is consistent with the DLS result. Therefore, the polypseudorotaxane of L3-CB[7] was formed indeed. The formation of the polypseudorotaxane was further confirmed by asymmetric flow field flow fractionation (As4F). As4F belongs to a family of field flow fractionation techniques, which is a suitable separation method to characterize macromolecules and particles. The As4F apparatus was connected in-line to ultraviolet, differential refraction, and multiangle light-scattering detectors. By fitting the data, molecular weight and distribution of our polypseudorotaxane could be obtained. As shown in Figure 3b, after adding 1.0 equiv of CB[7] into L3, the peak of the sample shifted to the right, indicating that a larger species compared with L3 was formed. The molecular weight of the polypseudorotaxane was calculated to be 1.85 × 104 g/mol with a polydispersity of 1.18. Therefore, these results corroborated the formation of the polypseudorotaxane. How can we realize control over antibacterial activity using this polypseudorotaxane? We envisioned that cationic polymers, such as ε-poly-L-lysine hydrochlorides, all have two key points for antibacterial use. One is the positive charge to bind with the negatively charged surface of the bacteria. The other is the hydrophobic component to insert into the cell membrane to destroy the osmotic equilibrium and then kill the bacteria. By host−guest complexation, the positive charge and the hydrophobic component of the cationic polymers can be both complexed with CB[7]’s cavity, leading to the decrease of the antibacterial efficiency, and then the antibacterial activity may be controlled in this way. To confirm this assumption, we performed a series of antibacterial experiments upon Escherichia coli (E. coli) using L3 (50.0 μM) with different ratios of CB[7]. On the agar plates, they were incubated for 20 h at 37.0 °C. Interestingly, the bacterial colony-forming units were gradually increased as the ratio of CB[7] became higher (Figure 4) because more CB[7] could bind with more antibacterial components, leading to more surviving bacterial colonies. Therefore, we eventually achieved control over antibacterial activity upon E. coli. Quantitatively, the inhibition ratios were further calculated according to the control group. As shown in Figure 5, the antibacterial efficiency could be well tuned. There was an
According to the 1H NMR and ITC experiments, a plausible binding model in the complexation is proposed as follows. As shown in Figure 2, three lysine units can be divided into one
Figure 2. Plausible binding model of the complexation between CB[7] and L3.
recognition unit and two equal spacer units, which can result in two series of the peaks of protons. The reason behind this model can be described that CB[7] is a bulky and rigid macrocycle with electron-rich carbonyl rims, and there would exist strong steric hindrance and electrostatic repulsion if CB[7] was too close to each other, thus making the spacer units necessary for the binding. Therefore, considering that the CB[7] cavity can only bind with one lysine unit, both of the other two lysines should function as spacer units. Dynamic light scattering (DLS) was employed to characterize the formation of the polypseudorotaxane L3-CB[7] and its aggregation. As shown in Figure 3a, the count rate gradually increased from 5 kcps to 30 kcps with the addition of CB[7], while the size of the aggregation slightly increased from 3.6 nm (L3) to 4.2 nm (L3-CB[7]). This indicated that by adding CB[7] the number of the aggregations in the solution increased significantly, but the size did not. There can be a possible explanation behind this phenomenon: L3 as a typical polyelectrolyte formed small aggregates which contained dozens of ε-poly-L-lysine hydrochlorides; however, by adding CB[7] the aggregation could be destroyed to release ε-poly-Llysine hydrochlorides to form the polypseudorotaxane into bulk, and as a result, the count rates increased. The small change of the size could also be confirmed by diffusion-ordered NMR spectroscopy (DOSY). The diffusion coefficient of L3 and L3-CB[7] was calculated to be 7.5 × 10−11 m2/s and 6.2 ×
Figure 3. (a) DLS data obtained by gradually adding CB[7] into L3 in PBS buffer of pH 6.0 at 25.0 °C ([L3] = 3.0 mM in all cases.). (b) As-4F results obtained by the UV detector of L3 and L3-CB[7] (the peaks around 6.0 min were the void peaks arising from the change of elution modes). 1111
DOI: 10.1021/acsmacrolett.6b00568 ACS Macro Lett. 2016, 5, 1109−1113
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cationic polymers, and moreover, other macrocyclic hosts could be used to construct functional polypseudorotaxane. Therefore, this line of research will enrich the field of polypseudorotaxanes and cationic polymers with important advances toward precise antibacterial regulation.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.6b00568. Experimental procedures, 1H NMR spectra, and other experimental results, along with supporting figures (PDF)
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Figure 4. Antibacterial experiments to demonstrate controlled antibacterial activity by tuning the ratio of CB[7] in L3-xCB[7] from 0 to 5.0 equiv ([L3] = 50.0 μM in all cases).
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by National Natural Science Foundation of China (21434004, 21274076) and the National Basic Research Program of China (2013CB834502). We thank Dr. Jiang-Fei Xu, Dr. Rong Hu, Ms. Linghui Chen, and Mr. Yuchong Yang for experimental assistance and helpful discussion.
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Figure 5. Inhibition ratio in different ratios of CB[7] to L3 ([L3] = 50.0 μM in all cases).
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