Self-immolative polymers with potent and selective antibacterial activity by hydrophilic side chain grafting

Cansu Ergene and Edmund F. Palermo *
Department of Materials Science and Engineering, Rensselaer Polytechnic Institute, 110 8th St., Troy, New York 12180, USA. E-mail:

Received 20th June 2018 , Accepted 12th July 2018

First published on 12th July 2018

We report the first example of a self-immolative polymer that exerts potent antibacterial activity combined with relatively low hemolytic toxicity. In particular, self-immolative poly(benzyl ether)s bearing pendant cationic ammonium groups and grafted poly(ethylene glycol) chains in their side chains were prepared via post-polymerization thiol–ene chemistry. These functional polymers undergo sensitive and specific triggered depolymerization into small molecules upon exposure to a designed stimulus (in this example, fluoride ions cleave a silyl ether end cap). The molar composition of the resulting statistical copolymers varied from 0 to 100% PEG side chains. The average molar mass of the pendant PEG chains was either 800 or 2000 g mol−1. The antibacterial and hemolytic activities were evaluated as a function of copolymer composition. Strong bactericidal activity (low μg mL−1 MBC) was retained in the copolymers containing 25–50% PEG-800, whereas hemolytic toxicity monotonically decreased (up to HC50 >1000 μg mL−1) with increasing PEG content. PEG-2000 was far less effective; both the MBC and HC50 decreased to a comparable extent with increasing PEGylation. Overall, the best cell type selectivity index (HC50/MBC ∼ 28) was obtained for the copolymer containing ∼50% cysteamine and ∼50% PEG-800 side chains, as compared to the cationic homopolymer (HC50/MBC < 1). Thus, the systematic tuning of the PEG graft density and chain length effectively enhances the cell-type selectivity of these self-immolative polymers by orders of magnitude.


The alarmingly rapid proliferation of antibiotic-resistant bacterial infections, compounded by the continuously declining number of new antibiotic drug approvals, is a global health crisis. The urgency of this problem has led many researchers to seek new antibacterial agents.1–3 Host defense peptides (HDPs) are components of innate immunity, which exert antibacterial efficacy with minimal toxicity to host cells.4–6 Synthetic mimics of HDPs including β-peptides,7 peptoids,8 nylon-3 copolymers,9 polymethacrylates,10–12 polymethacrylamides,13 polycarbonates14–17 and polynorbornenes18–20 are designed to obtain the essential physiochemical features of the peptides: cationic charge, hydrophobicity, and short chain length.21–25 HDPs and their synthetic mimics are widely thought to exert a mechanism of action involving membrane disruption26,27 and are less likely to induce bacterial resistance as compared to conventional antibiotic drugs.28 However, HDPs are expensive to manufacture and are rapidly degraded by proteases in vivo,29 which motivates the continued development of biomimetic synthetic polymers.30–32

In contrast to the proteolytic instability of HDPs, vinyl-based synthetic antibacterial polymers do not degrade appreciably under physiological conditions. Their chemical stability may restrict their use in biomedical application due to their long-term toxicity in vivo, even if they are non-toxic in short-term in vitro studies.17,33 Thus, biodegradable antibacterial polymers such as polyesters,33,34 polycarbonates15,35 and even acetal networks36 are of increasingly great interest. These polymers are subject to cleavage at random sites along the backbone of the polymer chains, thus representing a passive degradation rate that is dictated by the chemical structure of the linkage and the solvent-accessibility of the microenvironment.37 In contrast to conventional biodegradable polymers, metastable “self-immolative” polymers (SIMPs) undergo triggered end-to-end depolymerization in response to a specific stimulus.38 Upon cleavage of a labile ω-end-cap, above the ceiling temperature Tc, the active species of depropagation is liberated and the chain will spontaneously unzip into its component monomers.39–42 This unique phenomenon provides marked signal amplification, as well as mechanical transduction of chemical signals, and unparalleled specificity to control the onset of chemical degradation (rather than simply tuning the passive release profile). SIMPs have been used in applications such as drug delivery,43–45 biosensors,46 microfluidics47 and dynamically recyclable plastics48,49 but were not utilized in antimicrobial platforms until very recently. In the context of the development of antibacterial materials, self-immolation provides a unique opportunity to trigger the conversion from a polymer to a small molecule, which dramatically alters the mechanical properties and solubility characteristics of the material, on demand. One may envision application in “smart” bio-responsive coatings that mediate a triggered release of biologically active small molecules into the surrounding media.

We recently reported the first example of a biocidal self-immolative polymer50 based on modifications of the poly(benzyl ether) (PBE) platform pioneered by Phillips and co-workers.51,52 The cationic PBEs bearing primary amine groups displayed rapid, broad-spectrum antibacterial activity and were readily depolymerized into small molecules upon introduction of a chemical “trigger”. However, these first-generation cationic PBEs are also highly toxic to red blood cells (RBCs). We hypothesized that the intense hydrophobic nature of the PBE backbone leads to the high hemolytic toxicity and limits their aqueous solubility. In this work, we present a simple PEGylation strategy to decrease the overall hydrophobicity of the PBEs and thus reduce their hemolytic toxicity, while retaining their antibacterial potency.

The classical approach to HDP-mimetic design involves optimization of two key features in a polymer structure: the cationic charge and the hydrophobicity. However, binary copolymers were found to contain both higher cationic charge density and higher hydrophobicity compared to the average HDP.53 It is clear that HDPs are not solely composed of cationic and hydrophobic residues, but also contain an abundance of neutral, hydrophilic groups. Consequently, ternary copolymer systems containing neutral, hydrophilic groups (hydroxyls,54,55 sugars,56,57 zwitterions,58 PEG59,60) have been studied. Incorporation of the third component played an important role in modulating the cell-type selectivity of a polymer by reducing its hemolytic toxicity while maintaining (or even improving) its antibacterial efficacy. Youngblood and co-workers reported61 ternary antimicrobial copolymers of hydrophobically quaternized 4-vinyl pyridine (4VP) with PEG methacrylate. Compared to the highly antibacterial and hemolytic quaternized PVP, the PEGylated copolymers exhibited lower hemolytic toxicity and they retained their antibacterial activity.61

In this paper, we modified cationic PBEs with varying contents of PEG grafts in the side chains to quantify the effects of reducing hydrophobicity on their antibacterial and hemolytic activities. Poly(benzyl ether)s with allyl side chains and silyl ether end-caps were synthesized based on modifications of the route recently described by Phillips and co-workers.51 Relative to the first-generation cationic PBEs, the PEGylated variants exhibited lower hemolytic toxicity while maintaining a comparable level of antibacterial potency. The best example showed a 28-fold selectivity toward E. coli over red blood cells, which is a remarkable improvement over our first-generation biocidal SIMPs, which gave a selectivity index of <1.

Results and discussion

Polymer synthesis

We prepared a library of PBEs with PEGylated and cationic side chains, in various copolymer ratios and with two different PEG chain lengths (Fig. 1). Briefly, anionic polymerization was carried out at −20 °C in THF, which is below the ceiling temperature of PBE (Tc ∼ 0 °C at 1 M), followed by end-capping with TBDMS-Cl to give the pre-polymer P0. The monomer[thin space (1/6-em)]:[thin space (1/6-em)]initiator ratio was fixed at 10[thin space (1/6-em)]:[thin space (1/6-em)]1 for all polymerizations to give short oligomers, because the previous work has shown this chain length regime to be the most promising for nontoxic antibacterials.
image file: c8tb01632a-f1.tif
Fig. 1 General scheme for the synthesis of self-immolative antibacterial polymers with pendant PEG and ammonium groups: low-temperature anionic polymerization of a quinone methide monomer, end-capping, post-polymerization thiol–ene functionalization, and chemically triggered depolymerization with fluoride.

We obtained PBEs with the number-average molecular weights in the range of Mn = 3.4–3.6 kDa with dispersities of Đ = 1.42–1.57 by GPC. The moderately broad dispersities observed here are comparable to those observed in the previous reports on related polymerizations by Phillips51 and our group.50 Although the “livingness” of this polymerization has not been examined mechanistically to date, there are two reasonable hypotheses regarding the cause of the MWD broadness; either chain transfer or equilibration of the propagation and depropagation processes following a complete conversion. Similar the Mn values were found by 1H NMR end-group analysis (see the ESI). The side chains of P0 are allyl-functionalized for further modification.

The goal of this study was to incorporate neutral, hydrophilic functionality into self-immolative polymers to confer antibacterial activity with minimal hemolytic toxicity. To that end, the allyl side chains of the pre-polymer P0 were functionalized with cysteamine HCl (the thiol obtained by cleavage of the disulfide bond in cysteamine HCl) and PEG methyl ether thiol (PEG-SH Mn ∼800 Da or 2 kDa) via thiol–ene radical addition. The UV photoinitiator, Irgacure 651, was employed under irradiation of a handheld UV lamp (6 W, 365 nm) for 10 min at room temperature. In order to control the ratio of PEG and primary amines in the side chains, we first functionalized a fraction of the allyl side groups with PEG thiols, confirmed the partial conversion by 1H NMR (see data in the ESI), and then proceeded to functionalize the remaining unreacted allyl groups with excess cysteamine HCl. Each copolymer was purified by preparative size exclusion chromatography on LH-20 gel in MeOH. The copolymer compositions and the number-average chain lengths were quantified by 1H NMR for the final copolymer products (Fig. 2). See the ESI for the spectra of all polymers. We successfully obtained graft copolymers across the full range of copolymer compositions (from 0 to 100 mol% PEG). In all the cases, the observed copolymer composition is in good agreement with the target values (data in the ESI).

image file: c8tb01632a-f2.tif
Fig. 2 1H NMR spectrum (CD3OD) of P1-25-PEG800. Copolymer composition and degree of polymerization are calculated based on the ratios of integrated peak areas.

All the functionalized copolymers dissolve in polar solvents such as MeOH, DMF and DMSO. Whereas the copolymers containing 0–33% PEG800 are not soluble in aqueous buffers, and the copolymers with 50% or more PEG800 in side chains demonstrated facile water solubility (up to 1 mg mL−1). This observation is consistent with the idea that PEGylation alleviates the intense hydrophobicity of the PBE scaffold. Each copolymer was serially diluted in 2-fold increments from a DMSO stock solution (20 mg mL−1) into aqueous buffers to give the final polymer concentrations in the μg mL−1 range, which is the desired range for our biological assays. As a control experiment, we confirmed that the residual amount of DMSO (maximum 5% v/v under assay conditions) had no significant effect on the bacterial cell viability or on the hemolytic activity.

Dye-labeled polymers

We also prepared PBEs end-capped with rhodamine NHS ester, in order to enable their observation by confocal fluorescence microscopy (Fig. 5A and B), as well as to aid in the quantification of the water–octanol partition coefficients [log[thin space (1/6-em)]P = log([polymer]octanol/[polymer]water)], a standard measure of hydrophobicity that is widely used as a metric in structure–activity relationship (SAR) studies. Positive log[thin space (1/6-em)]P values indicate a preference for solubility in octanol, whereas negative values indicate a preference for the water phase. These dye labeled polymers were functionalized with cysteamine and PEG-SH in various ratios (0, 50, and 100% PEG) by the same methods mentioned above. Our hypothesis was that PEGylation would reduce the overall hydrophobicity of the copolymers. Indeed, the log[thin space (1/6-em)]P values exhibit a marked dependence on the PEG fraction. The log[thin space (1/6-em)]P values for the copolymers containing 0, 50, and 100% PEG800 in the side chains are +0.26, −0.56, and −1.20, respectively (Fig. 3). Thus, the hydrophobicity trend is quite clear: an increase in the PEG content leads to a markedly increased hydrophilicity in the copolymers, which strongly supports the central hypothesis driving this work. Encouraged by this result, we proceeded to perform SAR studies on this class of polymers.
image file: c8tb01632a-f3.tif
Fig. 3 Water–octanol partition coefficients (log[thin space (1/6-em)]P) for the rhodamine-labeled polymers with 0, 50, and 100% PEG800 in the side chains.

Bactericidal activity

Here the minimum bactericidal concentration (MBC) is defined as the lowest polymer concentration required to induce at least a 3-log reduction in the number of viable E. coli cells in PBS buffer after 90 min incubation at 37 °C. We quantified the effect of the PEGylation extent, as well as the length of the pendant PEG chains, on the bactericidal activity (Table 1 and Fig. 4). The cationic homopolymer, bearing 100% primary ammonium side chains (P1-0), exhibited a potent bactericidal action against E. coli with an MBC of 12 μg mL−1, which is similar to the MBC of the bee venom toxin peptide melittin (MBC = 4 μg mL−1) under the same assay conditions. In our earlier study, we reported similar polymers as strong bactericides with a high degree of cationic charge and hydrophobicity via a surfactant-like mode of action.
Table 1 Summary of the antimicrobial and hemolytic activities of cationic amphiphilic poly(benzyl ether)s as a function of molar percent PEGylation and PEG chain length
Polymer mol% PEG DPa MBC E. coli (μg mL−1) HC50 (μg mL−1) HC50/MBC
a Degree of polymerization (DP) by 1H NMR end group analysis. b Data from ref. 50 for comparison.
P1-100-PEG800 100 10 >1000 >1000
P1-85-PEG800 85 12 >1000 >1000
P1-63-PEG800 63 11 >1000 >1000
P1-57-PEG800 57 11 219 >1000 >4.6
P1-50-PEG800 50 12 12 340 28.3
P1-33-PEG800 33 12 12 83 6.9
P1-25-PEG800 25 12 26 89 3.4
P1-11-PEG800 11 12 26 25 0.96
P1-100-PEG2k 100 13 >1000 >1000
P1-70-PEG2k 70 13 >1000 >1000
P1-50-PEG2k 50 11 750 >1000 >1.3
P1-34-PEG2k 34 12 438 >1000 >2.3
P1-22-PEG2k 22 12 313 234 0.8
P1-12-PEG2k 12 11 94 46 0.5
P1-0 0 13 12 <8 <0.7
M2 100 >1000 >1000
M1 0 31 62 2
DP1-50-PEG800 (+ CsF) 50 8 104 13

image file: c8tb01632a-f4.tif
Fig. 4 Antibacterial activity and hemolytic activities of the polymers with varying mole % of (A) PEG-800 and (B) PEG-2k, as a function of PEG content.

To dilute the high degree of cationic charge and partially screen the hydrophobicity of the PBE backbone, we grafted hydrophilic PEG chains onto a fraction of the PBE side chains. Incorporation of modest amounts of PEG800 (11 and 25 mol%) did not significantly alter the antibacterial potency, with MBC values of 26 μg mL−1. A further increase the PEG content (33 and 50 mol%) resulted in MBC values of 12 μg mL−1, which is unchanged relative to the cationic homopolymer. Although PEGylation reduces the hydrophobicity and cationic charge of the copolymers, the improved aqueous solubility of the PEGylated copolymers is a countervailing effect that may offset the reduced electrostatic interaction with bacterial membranes.62 A further increase in the PEG800 content (beyond 50%) led to a dramatic loss of the antibacterial potency; the copolymer with 57 mol% PEG800 showed a modest MBC value of 219 μg mL−1 and those with 63 mol% or higher did not kill E. coli measurably even at the highest concentration tested (1000 μg mL−1).

The size effect of the grafted PEG chains on the antibacterial activity is rather pronounced. When PEG2k is employed instead of PEG800, the antibacterial potency decreases monotonically with increasing PEG content across the entire range of copolymer compositions (0–100%). Inclusion of just 12 mol% PEG2k in the side chains caused an increase in the MBC to 94 μg mL−1, compared to the cationic homopolymer MBC of 12 μg mL−1 (∼8-fold change). A further increase in the PEG2k content significantly abrogated the antibacterial activity, leading to MBC values on the order of some hundreds of μg mL−1. The copolymers containing 50 mol% or more PEG2k are completely inactive against E. coli even at the highest concentration tested (1000 μg mL−1). The simplest explanation for this trend is that the longer PEG2k chains excessively shield the cationic charge and the backbone hydrophobicity of the polymers, thus deterring the interaction between the polymers and the cell membranes to a greater extent than the shorter PEG800 chains.

It is widely understood that “amphiphilic balance” – finely tuning the interplay of cationic charge to a hydrophobic character – is central to the design rationale for antibacterial polymers.63,64 Overall, the results here show that PEGylation is an effective strategy to influence the “amphiphilic balance” of cationic, amphiphilic polymers, although judiciously tuning the PEG content and PEG chain length is clearly required. This stands in accordance with the literature precedent on other polymer platforms.61,62 The optimal formulation identified in this work is the copolymer containing ∼50% PEG800, which shows improved solubility and slightly enhanced antibacterial activity relative to the cationic PBE homopolymer (P1-0).

We also compared the activity of the dye labeled polymers to that of their unlabeled (silyl ether end-capped) counterparts. The MBC values for RhB-P1-0, RhB-P1-40-PEG800 and RhB-P1-100-PEG800 are 31, 31 and >1000 μg mL−1, respectively, which are comparable to those of P1-0, P1-50-PEG800 and P1-100-PEG800 (12, 12, and >1000 μg mL−1). Thus we conclude that the dye itself has only a marginal effect on the antibacterial activity, differing only by a single 2-fold dilution, and therefore these dye-capped polymers are appropriately representative of the polymer library in this work.

Hemolytic activity

The toxicity of these copolymers against mammalian cell membranes is quantified in terms of hemoglobin release from sheep red blood cells (RBCs). Here the hemolytic concentration (HC50) is defined as the characteristic polymer concentration that induces 50% hemolysis after 1 h incubation at 37 °C, as determined by curve fitting to the Hill equation. The cationic homopolymer (P1-0) was markedly hemolytic with an HC50 value lower than 8 μg mL−1, which is comparable to melittin (HC50 = 6 μg mL−1) under the same assay conditions, in agreement with our recent report.50 Incorporation of increasing amounts of PEG800 monotonically increases the HC50 values by orders of magnitude (Table 1 and Fig. 4). Addition of just 11 mol% PEG800 in the side chains reduced the hemolytic toxicity by ∼4-fold (HC50 = 25 μg mL−1). A further increase in the PEG800 content to 25% and 33% mole led to an additional ∼3-fold increase in the HC50 value (89 μg mL−1). This trend continued for the copolymers containing up to 50 mol% PEG800 (HC50 = 340 μg mL−1). A higher PEG800 content led to a complete loss of hemolytic activity with HC50 values above the highest concentration tested (1000 μg mL−1). Cell-type selectivity against bacterial cells without toxicity to mammalian cells is crucial for biomedical-related applications. Comparing the relative magnitudes of the HC50 and MBC values for the copolymers in this work reveals that the most promising example thus far is the PBE copolymer containing 50% PEG800 (HC50/MBC = 28.3). This metric represents a very marked improvement relative to our first-generation self-immolative antibacterial PBE (HC50/MBC = 0.5).50 Thus, we report the first example of a cell-type selective antibacterial polymer that possesses the unique self-immolative characteristic.

Incorporation of longer grafted PEG2k in the side chains exhibited a similar effect on the hemolytic activity; higher degrees of PEG substitution are associated with the loss of hemolytic toxicity in a monotonic manner. The slope of the HC50versus PEG mol% plot (Fig. 4) is higher for PEG2k than for PEG800, implying that the longer PEG chains impact the activity more significantly in the low PEG content regime. For example, incorporation of 12 mol% PEG2k gave an HC50 value of 46 μg mL−1, which is about 6-fold higher than the cationic homopolymer P1-0 and about 2-fold less hemolytic than the copolymer containing about the same molar percent of PEG800. An increase in the PEG2k mole content to 20% further decreased the hemolytic activity. All the polymers having 34% or more PEG2k in the side chains were non-hemolytic even at the maximum concentration tested, 1000 μg mL−1. By comparing two polymers with similar % mole of PEG, it is obvious that those with longer PEG2k units showed much more hemocompatibility relative to a polymer bearing shorter PEG800. For example, P1-34-PEG2k has an HC50 > 1000 μg mL−1, compared to P1-33-PEG800, which has HC50 = 83 μg mL−1 (a difference of more than 12-fold). The effect again can be explained by backbone screening by the longer grafted PEG chains, which shield the potent hydrophobicity of the benzyl ether backbone, thus diminishing the interactions with RBC lipid bilayers. Even though the polymers with longer PEG side groups are non-hemolytic, they are also less potent antibacterial agents, and hence do not exhibit excellent selectivity. The best cell-selectivity for a copolymer bearing PEG2k was a modest HC50/MBC > 2.3 for the polymer with 34 mol% PEG2k. This value is far lower than the best example from the PEG800 series, which was HC50/MBC = 28.3. It is thus reasonable to speculate that even shorter PEGs may have an even greater effect on the enhancement of the selectivity, although at some point the palliative effect is expected to diminish.

In contrast to negatively charged bacterial cell surfaces, the outer leaflet of the phospholipid bilayer in the RBC membrane displays a lower density of anionic charges.6,26 The hemolytic behavior of amphiphilic polymers has thus been mostly associated with their hydrophobicity, which results in membrane binding and surfactant-like disruption.65 The incorporation of hydrophilic PEG side chains plays a prominent role in dictating the hemolytic activity of synthetic polymers. Plasma proteins in blood get absorbed on RBCs and protect them from external stresses. In the absence of blood plasma, RBCs become more delicate and susceptible to lytic agents (such as in PBS, the assay media used here).66 PEG also behaves as a protective agent by shielding cells from foreign body contact. It has been shown that PEG gets weakly absorbed on the cell membrane via hydrogen bonding to improve protection through cells.67 Consequently, the hemolytic toxicity of the polymers containing PEG may also reduce by these or related mechanisms, in addition to its role in reducing the overall hydrophobicity of the copolymers.

Broad spectrum and kinetics

The most promising candidate identified in our initial screen was the copolymer containing 50 mol% PEG800 and 50 mol% cysteamine in the side chains, P1-50-PEG800. We further examined the bactericidal profile of this select formulation in terms of broad-spectrum activity and bactericidal kinetics (Fig. 5). The copolymer does exert bactericidal activity against both Gram-positive and Gram-negative bacteria. Bactericidal activity is generally more potent in the case of Gram-negatives; the MBC values against E. coli and P. aureginosa are 12 and 4 μg mL−1, respectively, whereas against S. aureus and E. faecalis, the values are 250 and 125 μg mL−1. This observation is similar to our previous findings with PBE cationic homopolymers, which exerted much more potent activity against Gram-negatives compared to Gram-positives. In addition, it was found that the kinetics are slower against Gram-positives, requiring 4 h incubation to induce a 4-log reduction in the number of viable S. aureus cells (99.99% killing) as compared to less than 1 h in the case of E. coli. It is interesting that these PEGylated PBEs generally show better activity against Gram negative strains relative to Gram positives. Indeed, many examples of antibacterial polymers are more active against Gram-positive bacteria relative to Gram-negatives, although there are also examples that show the opposite preference.68 Lienkamp et al., in a study, found that there may also be a molecular sieving effect of the peptidoglycan layer in Gram positives.69 The details of the mechanism of action are outside the scope of this work, but we hypothesize that the thick, cross-linked peptidoglycan layer present in Gram positives may be difficult for these PBE-graft-PEG copolymers to translocate.
image file: c8tb01632a-f5.tif
Fig. 5 (A) Bactericidal kinetics of P1-0 and P1-50-PEG800 against E. coli in PBS at 2 × MBC and (B) broad spectrum activity against Gram-positive and -negative bacteria.

The PEGylated copolymer P1-50-PEG800 exerts markedly faster bactericidal kinetics against E. coli (4-log reduction in <30 min) relative to the cationic homopolymer P1-0 (∼90 min), as shown in Fig. 4A. This observation initially seems counterintuitive because the PEGylated copolymer has a lower cationic charge density and lower hydrophobicity, relative to the cationic homopolymer, and the membrane disruption is thought to depend on both electrostatic and hydrophobic interactions. We hypothesized that the PEGylated polymer acts faster overall due to the more subtle hydrophobicity, which disfavors aggregation in solution and thus may facilitate the binding of individually solvated polymer chains onto the bacterial cell envelope. In contrast, the excessively hydrophobic P1-0 may exist as aggregates in solution that display cationic groups on the surface and bury the hydrophobic residues in an inner globule. This sort of hydrophobic clustering hypothesis has been invoked to explain the reduced antibacterial activity in the case of excessively hydrophobic polymethacrylates.70

To probe our hypothesis, we used the rhodamine-labeled variants of P1-0, P1-50-PEG800, and P1-100-PEG800 each containing one dye tag in their terminal end group (see the ESI for synthetic details). The MBC values of these tagged polymers are similar to those of the untagged polymers of the same copolymer composition (data in ESI). Indeed, confocal microscopy images of RhB-P1-0 show micron-sized aggregates in PBS buffer at concentrations as low as 16 μg mL−1 (Fig. 6A), which is similar to the MBC value. In stark contrast, RhB-P1-50-PEG800 and RhB-P1-100-PEG800 both show a diffuse, uniform fluorescence intensity in PBS even up to 1000 μg mL−1, which is well above the MBC value (Fig. 6B). A detailed study on the polymer aggregates using Dynamic Light Scattering (DLS) is beyond the scope of this work, but may be an interesting avenue for future work.

image file: c8tb01632a-f6.tif
Fig. 6 Confocal images of the (A and B) rhodamine dye-labeled polymers in PBS media and the (C and D) non-labeled polymers at 2× MBC with E. coli and LIVE/DEAD stains.

Moreover, when the rhodamine-labeled polymers are mixed with E. coli cells, there is a very clear localization of RhB-P1-50-PEG800 specifically on the cell membrane, as observed from the confocal images (Fig. 7A). In contrast, when RhB-P1-0 is mixed with E. coli cells, aggregates on the order of 10–30 microns are observed, with no clear individual rod-shaped bacterial cells (Fig. 7B). These data strongly support the notion that the activity of the cationic homopolymer P1-0 is indeed hampered by the extensive polymer–polymer aggregation in the aqueous media, as we expected. The PEGylation strategy indeed alleviates the excessive hydrophobicity of the PBE backbone and brings the global physiochemical properties of the copolymer into the appropriate range for “amphiphilic balance” required to obtain favorable biological activity.

image file: c8tb01632a-f7.tif
Fig. 7 (A) Rhodamine-labeled polymer with 50% PEG800 in the side chains binds specifically to the membranes of individual planktonic E. coli cells, seen as bright emission from the outlines of the smooth rod-shaped cells. (B) Rhodamine-labeled polymer with no PEG (all cysteamine side chains) shows only indistinct aggregates.

Triggered depolymerization

We next examined the influence of specifically triggered depolymerization on the antibacterial and hemolytic activities of the PEGylated cationic PBEs. In accordance with our prior work, TBDMS-capped pre-polymers (P0) undergo rapid depolymerization into small molecules upon fluoride exposure, as evidenced by 1H NMR and GPC.50 In this work, fluoride was introduced as an exogenous trigger. In general, the PBE platform boasts a great deal of diversity in terms of end-cap/trigger combinations that can be used to mediate self-immolation. The present example was selected for synthetic accessibility and because the fluoride ion is biologically inactive against bacterial and human cells. Indeed, we are also interested in expanding this work to include naturally occurring endogenous triggering chemistries, perhaps involving natural changes in redox, pH, or enzymatic cues, but these efforts are outside the scope of the present work.

Triggered depolymerization of P1-50-PEG800 was initiated using cesium fluoride (CsF) in MeOH or DMF, and monitored by 1H NMR and GPC (Fig. 8). Upon F treatment, the broad features in the 1H NMR spectra attributed to the PBE backbone disappeared and were replaced by sharp peaks corresponding to the small molecule products of depolymerization. The disappearance of the resonance at ∼5.5 ppm is used to quantify the percentage of depolymerization, which reached completion after stirring overnight (Fig. S35, ESI). The GPC trace (in DMF) also shows a corresponding shift to longer retention times, in accordance with the products of a smaller hydrodynamic volume that match the chromatogram for the PEG800 grafted monomer M2. The MBC for the intact polymer P1-50-PEG800 was 12 μg mL−1. In response to fluoride, P1-50-PEG800 degraded into its small molecule components (referred to as DP1-50-PEG800 in Table 1), which had an MBC value of 8 μg mL−1. Thus, the products of depolymerization appeared to exert a similar antibacterial activity to that of the intact polymer. The small change in the MBC upon depolymerization is consistent with the observation that the small molecule product of depolymerization containing an ammonium cation is a potent antibacterial agent, although the PEGylated monomer shows no antibacterial activity.

image file: c8tb01632a-f8.tif
Fig. 8 1H NMR (CDOD3) and GPC (DMF) of P1-50-PEG800 before and after initiation using CsF in MeOH, with its corresponding chemical structures and biological activities.

We also investigated the biological activities of model small molecule compounds that contain either the primary amine (M1 in Table 1) or PEG800 (M2) attached to a single unit of bisphenol (chemical structures given in Fig. 1) as a standard for comparison. In contrast to the potent antibacterial activity of the primary amine functionalized monomer M1 (31 μg mL−1), the PEGylated monomer M2 was inactive against E. coli up to 1000 μg mL−1.

Triggered self-immolation also influences the hemolytic activity compared to the intact polymer. For example, P1-50-PEG800 (HC50 = 340 μg mL−1) depolymerized into DP1-50-PEG800 (HC50 = 104 μg mL−1), which is about a 3-fold change. This result can be understood in terms of the HC50 values for the two monomer components: 62 μg mL−1 for M1 and >1000 μg mL−1 for M2. If one considers that the two are present in approximately a 1[thin space (1/6-em)]:[thin space (1/6-em)]1 ratio (for this 50–50% copolymer), and that one component is completely inactive, then we expect the simple mixture to have an HC50 value that is simply twice as that of the active component (2 × 62 = 124 μg mL−1). The experimental result is 104 μg mL−1, which is in reasonably good agreement with the prediction for a simple mixture. The slight differences may be due to the presence of some amounts of a dimeric species, which has been observed previously in the depolymerization byproducts for PBE.50

In addition to the MBC assay used to quantify cell death in terms of the number of viable colonies observable to the unaided eye, we also evaluated the antibacterial activity of the polymers and their small components by confocal laser scanning microscopy to investigate the antibacterial effect at the individual cell level, before and after depolymerization (Fig. 9).

image file: c8tb01632a-f9.tif
Fig. 9 Confocal images of the E. coli cells (a–c) alone, (d–f) after exposure to P1-50-PEG800, (g–i) after exposure to CsF-depolymerization product DP1-50-PEG800. Scale bar is 10 μm in all images. Additional images of the cells treated with M1 and M2 are given in the ESI.

For this study, we employed the commercially-available BacLight LIVE/DEAD staining kit, which includes the proprietary SYTO 9 dye (green) that stains both live and dead cells indiscriminately, and propidium iodide (PI, red) that emits only when intercalated into DNA within the cytoplasm.71 PI staining is thus used as a metric to confirm the permeabilization of the cell membrane, which is the putative mechanism of action for cationic, amphiphilic antibacterial agents.72–76 Firstly, as a positive control, E. coli cells in the absence of polymers were examined. In the confocal images, we observed smooth, rod-shaped E. coli cells in the green SYTO9 channel with no detectable emission in the red PI channel, which suggests healthy live cells (Fig. 9a–c). To assess the impact of the polymers, E. coli (5 × 107 CFU mL−1) was incubated for 90 min at 37 °C in the presence of antibacterial polymers above their lethal concentration (4 × MBC). We examined both the intact copolymer P1-50-PEG800 (Fig. 9d–f) as well as the product of the triggered depolymerization DP1-50-PEG800 (Fig. 9g–i). Both samples showed bright emission in the green and red channels, suggesting the substantial co-localization of both fluorophores within the cells.

Based on the fact that the PI stain cannot translocate healthy cell membranes, combined with a specific membrane stained using our Rh-labeled polymers, we conclude that the DNA-intercalated PI stain within the cytoplasm is associated with a deterioration of the cell membrane barrier function upon exposure to the antibacterial polymers. Thus, it would appear that the polymers in this work are capable of permeabilizing the E. coli cells, in accordance with numerous other literature examples on cationic, amphiphilic polymers.72–76

The primary amine functionalized model monomer M1 also showed extensive PI staining within the cells (images in the ESI). On the other hand, the monomer with PEG800 functionality M2 did not induce any red PI emission from cells, which again confirms that the PEG800 monomer units are not bactericidal (images in the ESI). Interestingly, the confocal images in this work are rather different in appearance relative to our previous report on the cationic homopolymer. Whereas P1-0 showed red PI staining of large cell–cell aggregates on the order of several tens of microns (Fig. 6C),50 the PEGylated variants in the present study appear to stain cells individually without inducing extensive cell–cell aggregation (Fig. 6D and 7B), which is also consistent with the concept of reduced hydrophobicity (more negative log[thin space (1/6-em)]P values) for the PEGylated polymers.

Control experiments

The CsF was not removed prior to testing the biological activity; CsF showed no effect on the E. coli cell viability even at concentrations 10× higher than those present in the assays. As a negative control for triggering, PBEs were end-capped with inert methyl groups, side-chain functionalized and exposed to CsF under the same conditions. Fluoride-triggered depolymerization did not occur in these polymers, as evidenced by 1H NMR, and tested for MBC and HC50. Before and after CsF exposure, MBC and HC50 values did not show any significant changes. Hence, we confirmed that fluoride-triggered depolymerization is specific to silyl ether end-capped polymers. In addition, we confirmed that no adverse side reactions occur when the allyl functionalized pre-polymer P0 is exposed to cysteamine (or PEG-SH) under UV irradiation with no photoinitiator present. Similarly, no reaction is observed when P0 is exposed to the photoinitiator under UV irradiation without any thiol present.


Materials and methods

Reagents were purchased commercially and used as received without further purification unless otherwise noted. 4,4′-Methylenebis(2,6-dimethylphenol) was purchased from Tokyo Chemical Industry (TCI America, USA). Potassium carbonate (K2CO3), allyl bromide (allyl Br), ammonium chloride (NH4Cl), sodium chloride (NaCl), anhydrous sodium sulfate (Na2SO4), silver oxide (Ag2O), tert-butyldimethylsilyl chloride (TBDMS-Cl), imidazole, 2-aminoethanethiol hydrochloride (cysteamine), poly(ethylene glycol) methyl ether thiol (Mn = 800 g mol−1 and Mn = 2000 g mol−1), 2,2-dimethoxy-2-phenylacetophenone (DMPA), cesium fluoride (CsF), Triton X-100, sodium phosphate monobasic monohydrate and sodium phosphate dibasic heptahydrate were purchased from Sigma-Aldrich (USA). 1-tert-Butyl-2,2,4,4,4-pentakis(dimethylamino)-2λ5,4λ5-catenadi(phosphazene) (P2-t-Bu base) (2.0 M solution in THF) was also purchased from Sigma-Aldrich and stored in a glove box under a N2 atmosphere. BacLight™ Bacterial Viability Kit L-7007 and NHS-rhodamine (5/6-carboxy-tetramethyl-rhodamine succinimidyl ester) were purchased from Thermo Fisher Scientific (USA). 10% (v/v) red blood cells (RBCs) were obtained from MP Biomedicals (USA). Organic solvents diethyl ether (Et2O), N,N-dimethylformamide (DMF), ethyl acetate, hexane, methanol (MeOH), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (USA). Isopropanol (iPrOH) was distilled before use. Anhydrous tetrahydrofuran (THF) was obtained from a solvent purification system. Deionized water was purified using an EMD Millipore purification system. Sephadex LH-20 was obtained from Sigma-Aldrich. Flash-column chromatography was employed using silica gel (60 Å pore size, 40–63 μm technical grade, Sigma-Aldrich). Thin-layer chromatography was performed on IB2-F J.T. Baker silica gel TLC (Germany).

Proton nuclear magnetic resonance (1H NMR) spectra were recorded using a 500 MHz Agilent NMR spectrometer at 25 °C. The NMR chemical shifts are reported in parts per million (ppm, δ) and referenced to tetramethylsilane ((CH3)4Si, 0.00 ppm) or to residual solvent signals (CDCl3 (δ 7.27), (CD3)2OS (δ 2.50), or CD3OD (δ 3.31 and 4.78)). Carbon nuclear magnetic resonance (13C NMR) spectra were recorded using a 500 MHz Agilent NMR spectrometer at 25 °C. The NMR chemical shifts are reported in parts per million (ppm, δ) and referenced to residual solvent signals (CDCl3 (δ 77.0), or CD3OD (δ 49.0)).

Size exclusion chromatography (SEC) was performed on an Agilent Technologies 1260 Infinity GPC system equipped with a refractive index detector and PLGel columns using THF and DMF as the mobile phase (flow rate: 1 mL min−1, 25 °C for THF, flow rate: 1 mL min−1, 45 °C for DMF). The molecular weight was calibrated using monodisperse polystyrene standards.

Laser scanning confocal microscopy (Zeiss LSM 510 Meta) was performed using argon (458–488–514 nm) and HeNe1 (543 nm) lasers. 512 × 512 pixel images were recorded from a single scan.

Mass spectra were measured on a Thermo LTQ XL Orbitrap mass spectrometer (Thermo, Bremen, Germany) with an electrospray ionization ion source. Samples were injected using an Agilent 1200 nano-HPLC system (Agilent, Palo Alto, CA) using an Agilent 1200 autosampler. The flow rate of the solvent was 50 μL min−1. The injection volume was 1–2 μL. The data were collected in the m/z range of 100–900 at a resolution of 30[thin space (1/6-em)]000. The accuracy of the mass measurements was ∼3 ppm.

Monomer synthesis

Allyl Br (1.0 equiv.) was added drop-wise to a stirred mixture of 4,4′-methylenebis(2,6-dimethylphenol) (1.0 equiv.) and K2CO3 (1.1 equiv.) in DMF (0.4 M). After 24 h reaction at room temperature, the mixture was extracted with ethyl acetate and deionized H2O. The organic layer was washed with a saturated NH4Cl solution and then with brine. It was dried over anhydrous Na2SO4, filtered to separate salts and concentrated via rotary evaporation. On the TLC plate, three spots were observed, which are assigned to the compounds with double allyl and single allyl in the side chains, and the unreacted starting material. The viscous oil was purified by silica gel column chromatography with gradient elution of solvents (10–33% ethyl acetate in hexanes) to afford a compound with single allyl as a yellow oil (yield 28%).

Ag2O (2.0 equiv.) was added into the solution of the single allyl compound (1.0 equiv.) and Et2O (0.1 M). The reaction mixture was stirred for 16 h at room temperature. The mixture was filtered to remove silver oxide particles, concentrated using a rotary evaporator and recrystallized in hot cyclohexane to afford yellow crystals. The monomer M0 crystals were ground and dried under vacuum for 72 h. The dry monomer (67%) was stored in an inert glove box atmosphere.

Polymer synthesis

The monomer M0 (1.0 equiv.) was dissolved in anhydrous THF. A stock solution of distilled iPrOH (0.1 equiv.) and a 2.0 M P2-t-Bu base solution (0.1 equiv.) in THF was prepared in anhydrous THF (1[thin space (1/6-em)]:[thin space (1/6-em)]1 ratio). The chain length of the polymer was tuned by altering the number of equivalents of initiator relative to the monomer. Based on this, a certain amount of the initiator-base stock solution was added into a monomer solution to pre-initiate and stirred for 1 h at room temperature. The final concentration of the reaction mixture was adjusted to 0.8 M. The reaction mixture became dark red from bright yellow color after the addition of base. Following the initiation, polymerization was conducted in −20 °C for 4 h under stirring. All the steps were carried out in a glove box under an inert N2 environment.


TBDMS-Cl (1.0 equiv.) and imidazole (1.0 equiv.) were dissolved in anhydrous THF and then injected into the polymer reaction mixture at −20 °C, which immediately turned into orange-yellow color from dark red. The reaction mixture was stirred for 24 h at −20 °C. It was allowed to warm to room temperature and the stirring was continued at this temperature for a few hours. The polymer was precipitated in MeOH and collected via a centrifuge. Excess MeOH was decanted. The polymer was redissolved in THF and precipitated in MeOH and centrifuged again, and the whole process was repeated three times. The polymers were dried under vacuum for 24 h. The yields of each polymerization are given in the ESI.

The synthetic procedures largely followed the precedent by Phillips,51 and our own prior report,50 with minor modifications. Further synthetic details, triggered depolymerization, NMR and GPC data, biological assay protocols, and microscopy procedures, are included in the ESI document.


We functionalized the side chains of a self-immolative poly(benzyl ether) platform with a combination of cationic ammonium groups and neutral, hydrophilic PEG chains. The antibacterial and hemolytic activities of these copolymers are sensitively dependent on the cationic charge and the hydrophobicity of their polymer chains. Here, we find that side chain PEGylation is a highly effective strategy to modulate their hydrophobicity and to enhance their cell-type selectivity. The copolymer composition (molar percentage of PEG grafts) and the PEG chain length are the key design parameters for tuning their biological activity. From our library of PBE copolymers, the most promising example is the one that contains 50 mol% PEG800 and 50 mol% primary ammonium cationic side chains. This select example exerts excellent 28-fold selectivity for bacterial cells relative to mammalian RBCs. The sensitive and specific self-immolative characteristics of the backbone are retained upon side chain functionalization.

In this work, we used stimuli-responsive end-caps that are cleaved on demand by an externally applied stimulus, like fluoride ions. The beauty of the self-immolative PBE platform is that these polymers can be designed with a variety of responsive groups, which are triggered by light, pH, or redox. Currently, we are developing biologically active degradable macromolecules that can be degraded by naturally occurring stimuli including bacterial enzymes. We envision that these materials may be useful in a broad range of antibacterial and antibiofilm coatings with unique “triggered-cleaning” characteristics. These and related studies are currently underway in our laboratory.

Conflicts of interest

There are no conflicts to declare.


We thank Prof. Scott Phillips for helpful discussions on self-immolative polymer synthesis, Prof. Sangwoo Lee and Prof. Chulsung Bae for their help in use of GPC, Dr Ao Chen for helpful discussion, and undergraduate students Samuel Ellman, Sadjo Sidikou, and Phillip Falcone for their assistance with the synthesis and scale-up of the monomers used in this study. E. F. P. acknowledges funding from a 3M Non-Tenured Faculty Award, a National Science Foundation CAREER Award (DMR BMAT #1653418), and the American Chemical Society Petroleum Research Fund (57806-DNI7). C. E. was supported in part by a Presidential Graduate Research Fellowship from the Rensselaer Polytechnic Institute.


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Electronic supplementary information (ESI) available. See DOI: 10.1039/c8tb01632a

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