Switchable Control of Antibiotic Activity: A Shape-Shifting “Tail

Dec 12, 2017 - Given a signature fluorine substituent in ciprofloxacin, X-ray photoelectron spectroscopy (XPS) was also employed to investigate the ch...
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Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

Switchable Control of Antibiotic Activity: A Shape-Shifting “Tail” Strategy Jinming Chang,† Yi Chen,*,†,‡ Zhou Xu,† Zhonghui Wang,† Qi Zeng,† and Haojun Fan† †

Key Laboratory of Leather Chemistry and Engineering of Ministry of Education, Sichuan University, Chengdu, 610065, P.R. China Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States

‡

S Supporting Information *

ABSTRACT: Bacterial resistance is emerging as a global threat, stemming partially from continuous exposure of pathogens to antibiotics of sublethal concentrations. Thus, novel molecular approaches capable of inactivating antibiotics, which prevent their final build-up in the environment, are highly desirable. Here, we report a proof-of-principle demonstration of a mechanically new strategy for switchable control of antibiotic activity, which regulates drug uptake across the outer membrane of Gram-negative bacteria by externally triggered shape shifting of a short, covalently attached “tail”. The rationale behind this strategy is grounded in the sizeselectivity of porin channels exploited by a large proportion of antibiotics for accessing intracellular targets, thus representing a general approach to control antibiotic availability in the environment which alleviates undue selection pressure for resistance.

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rejoining.12 To approach their intracellular targets, quinolones have to traverse the whole cell envelope, with the outer membrane (OM) being the first and major influx barrier in Gram-negative bacteria. Low permeability of OM against external noxious agents has been well understood, attributable to occupancy of its outer leaflet by a lipopolysaccharide monolayer of a highly order quasicrystalline structure.13 For nutrient acquisition, Gram-negative bacteria have evolved multiple trans-OM β-barrel proteins, or porins (e.g., OmpF, OmpC, OmpA in Escherichia coli), serving as nonspecific aqueous channels to allow influx of hydrophilic solutes. Since the late 1980s, a host of quinolone-resistant laboratory mutants and clinical isolates have been reported, which invariably showed complete or partial loss of OmpF.14−17 Thus, a porinmediated pathway has been recognized as the major route for quinolone uptake.18 Here, it is noteworthy that the porin channels possess a relatively well-defined effective aperture (ca. 0.7−1.2 nm in diameter), and thus, are size-selective.19 They only allow passage of hydrophilic molecules of a certain size, but exclude larger hydrophilic molecules or hydrophobic ones. This size selectivity leads us to envisage that switchable activity for quinolone (or other analogues exploiting porins for translocation) may be possible, if the drug molecules possess a convertible shape, allowing intracellular access via porins in one geometric form, while becoming too bulky to traverse these restrictive channels in the other. Of course, antibiotic molecules are not deformable themselves. Here, we hypothesize that attaching the drug

INTRODUCTION Antibiotic resistance is escalating at an unprecedented rate. As the current antibiotic pipeline diminishes, the crisis worsens, and now we find ourselves close to where we started-in the preantibiotic era. In general, the emergence and dissemination of resistant variants stem in part from antibiotic build-up in the environment, following their extensive use in antibiotherapy.1−3 Continuous exposure to antibiotics of sub-lethal concentrations encourages cellular mutagenesis and horizontal gene transfer, giving rise to “superbugs”, estimated to account for globally, around 700,000 deaths every year.4,5 Despite constant efforts to curtail inappropriate antibiotic prescription, another promising approach to counteract resistance emerged recently, which relies on unconventional molecular strategy that inactivates the drugs without physical disruption, thus avoiding their accumulation in the environment. One compelling example pertains to photodegradable β-lactam, which undergoes lightinduced destruction of its β-lactam moiety and hence becomes biologically inactive.6 Another level of control over antibiotic activity can be accomplished with photoisomerizable scaffolds that inactivate the conjugated antibiotic by switching from trans to cis isomers with light.7−9 Our understanding of how prototypical small-molecule antibiotics induce bacterial death sheds light on mechanistically new strategies for switchable activity. Although a complex process, antibiotic-mediated cell death invariably involves initial drug diffusion across biological barriers, followed by inhibiting a repertoire of targets essential for structural integrity or physiological functions of the cells. 10,11 For example, quinolones are a family of synthetic broad-spectrum antibiotics. They attack DNA gyrase and topoisomerase IV, trapping these enzymes at the DNA cleavage stage to prevent strand © XXXX American Chemical Society

Received: October 8, 2017 Revised: November 30, 2017

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DOI: 10.1021/acs.bioconjchem.7b00599 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry Table 1. Chemical Compositions of Cipro-PNIPAMn molecular weightsa of the PNIPAM “tail”

composition

conjugate

Mn (g/mol)

Mw (g/mol)

PDI

nNIPAMb

nCiprofloxacinb

ciprofloxacin contentc (mol %)

Cipro-PNIPAM23 Cipro-PNIPAM43 Cipro-PNIPAM72 Cipro-PNIPAM90

2592 4808 8179 10190

2757 5044 8588 10899

1.06 1.05 1.05 1.07

23 43 72 90

2.2 1.7 1.1 0.5

8.0 3.9 1.5 0.7

a Determined from size exclusion chromatography (SEC) in tetrahydrofuran (SI, Figure S3). bnNIPAM-average number of NIPAM monomeric units per conjugate chain; nCiprofloxacin-average number of N-acryloyl ciprofloxacin monomeric units per conjugate chain; both calculated from 1H NMR spectroscopies (SI, Figure S2). cObtained from pyrolysis-gas chromatography/mass spectrometry.

with a short, shape-shifting “tail” may address this challenge. As the “tail” fully stretches, displaying a small cross-section perpendicular to its length, the drug “head” may still have the chance to enter and finally diffuse through the OM-spanning porins, approaching specific targets for biocidal action. Once the “tail” deforms, for example, into a bulky entity, those restrictive porins appear too narrow to allow translocation of the whole conjugate, excluding the drug “head” from accessing intracellular targets. In this way, the engineered antibiotics may display appreciably different, or switchable, activity in response to the stimulus that triggers shape-shifting of the “tail”. To demonstrate this hypothesis, ciprofloxacin, a representative of the second-generation quinolones, was engineered herein, followed by covalent attachment of a short, thermally sensitive poly(N-isopropylacrylamide) (PNIPAM) “tail”. Once heated across the lower critical solution temperature (LCST), PNIPAM in water collapses from an extended random coil with a small cross-section perpendicular to its length, into a compact, bulky globule, interpreted as due to entropically driven hydrophobic association of nonpolar isopropyls.20−22 With this shape-shifting “tail”, we anticipated that the ciprofloxacin “head” was still bactericidal at low temperature, while being rendered inactive upon heating above the LCST. Temperaturedependent susceptibility to this conjugate proved relevant to differential drug uptake via those trans-OM, size-selective porins, dictated by geometric shape of the PNIPAM “tail”. Since a large proportion of antibiotics (e.g., chloramphenicol, tetracycline, and some β-lactams)23 exploit porin channels for internalization, the shape-shifting “tail” strategy proposed herein may be generalized to other systems, counteracting resistance by preventing accumulation of active drugs in the environment.

allowed a fast dynamic equilibrium to be established between the active propagating radicals and the dormant polymeric CTA compound, providing equal probability for all chain growth and, thus, a narrow polydispersity. Using mixtures of monomer, initiator, and RAFT agent of various compositions, a series of short, narrow-polydispersed (PDI ≤ 1.07) PNIPAM with different molecular weight was synthesized (Table 1). More importantly, retention of a thiocarbonylthio cap in the PNIPAM product enabled consecutive polymerization of vinyl ciprofloxacin monomers from only one end of the “tail”, yielding a lollipop-like conjugate. To this end, ciprofloxacin was acryloylated by site-specific reaction of the secondary amine in the 7-piperazinyl substitute with acryloyl chloride. Our recent investigations25,26 showed that the resultant N-acryloyl ciprofloxacin was still bioactive, because the 3-carboxyl, 4keto, and quinolone pharmacophore, all essential for hydrogenbonding with DNA gyrase and topoisomerase IV to elicit inhibitory action on in vivo DNA synthesis, remained intact. By consecutive RAFT polymerization, the PNIPAM “tail” was attached to the antibiotic “head” via noncleavable linkages, preventing artifacts by undesirable release of small-molecule antibiotics. The resultant conjugate is denoted as CiproPNIPAMn, where Cipro represents ciprofloxacin while the subscript n denotes the average number of NIPAM monomeric units per conjugate chain. From 1H NMR spectroscopies (SI, Figure S2), the average number of N-acryloyl ciprofloxacin monomeric units per conjugate chain varied from ca. 2.2 to 0.5 as the “tail” length increased, consistent with the results from pyrolysis-gas chromatography/mass spectrometry, indicating a decreased ciprofloxacin content from ca. 8.0 mol % in CiproPNIPAM23 to ca. 0.7 mol % in Cipro-PNIPAM90 (Table 1). Limited copolymerization of N-acryloyl ciprofloxacin was simply due to the steric hindrance imposed by bulky ciprofloxacin. Although short and covalently attached, the PNIPAM “tail” remained thermally sensitive, experiencing the well-known coilto-globule transition upon heating, macroscopically manifested as a sudden change of transmittance with increasing temperature, as recorded by turbidimetry. In comparison with pure PNIPAM products exhibiting a molecular weight-dependent LCST ranging from 29.8 to 32.0 °C (SI, Figure S4), single endcapping with a limited number of vinyl ciprofloxacin monomers slightly lowered the temperature where the coil-to-globule transition occurred, with the LCSTCipro‑PNIPAMn detected in the range of 28.9−31.2 °C (Figure 1). Using the 2-fold broth microdilution method, minimum inhibitory concentration (MIC) values of these ciprofloxacinbearing conjugates against Gram-negative Escherichia coli (E. coli) K12 (wild type) were determined at 25 °C, well below the LCSTCipro‑PNIPAMn, or at 33 °C, when the “tail” shape-shifted into a bulky globule. For comparison, the reported MICs were

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RESULTS AND DISCUSSION To attach ciprofloxacin to only one terminal of the PNIPAM “tail”, reversible addition−fragmentation chain-transfer (RAFT) polymerization technique, allowing chain extension of a polymer of one monomer with a second monomer, was employed to prepare the conjugate. The RAFT agent, 2(butylthiocarbonothioylthio)-2-methylpropanoate (CTA), was synthesized using a previously disclosed procedure.24 Chemical structure of the product was validated by comparing its Fourier transform infrared (FT-IR), 1H and 13C nuclear magnetic resonance (NMR) spectroscopies (SI, Figure S1) to those previously reported.24 Once initiated by 2,2′-azobis(2-methylpropionitrile) (AIBN), addition of a propagating PNIPAM radical to the RAFT agent, followed by fragmentation of the adduct, gave rise to a dormant polymeric CTA compound and a reinitiating radical. Reaction of this radical with residual Nisopropylacrylamide (NIPAM) monomers then yielded a new propagating radical. Subsequent addition−fragmentation steps B

DOI: 10.1021/acs.bioconjchem.7b00599 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

found higher than that against E. coli K12 by a factor of 8, while attaching a PNIPAM “tail” of 90 monomeric units only yielded a 2-fold increase in MIC against the OmpF-depleted phenotype. This phenomenon evinced that ciprofloxacin with a relatively shorter PNIPAM “tail” still preferred to take the porin channels for internalization, while a longer “tail” might force the conjugate to access the cells by hydrophobic pathway. Therefore, if the size selectivity of porins should be utilized to regulate ciprofloxacin internalization, the PNIPAM “tail” must be short enough such that other selectivity-destroying uptake pathway could be minimized. In comparison to the MICs at 25 °C (i.e., below LCSTCipro‑PNIPAMn), all conjugates displayed decreased activity against E. coli K12 at 33 °C, with the measured MICs doubled for Cipro-PNIPAM90, while displaying a 16-fold increase in the case of Cipro-PNIPAM23 (Table 2). In particular, the on−off ratio of Cipro-PNIPAM23 was found significantly improved beyond that of some light-switchable antibiotics previously reported (on−off ratios ranging from 2 to 4).7−9 Such pronounced temperature-dependent difference in susceptibility should be closely associated with geometric shape of the PNIPAM “tail”, which influenced transport of the ciprofloxacin “head” through the narrow porin channels (Scheme 1).

Figure 1. Plots of the changes in transmittance (%) as a function of temperature for aqueous solutions of Cipro-PNIPAMn. The sample concentration was 1.0 g/L, and the heating rate was 0.2 °C/min. The LCST was taken as the temperature of the solutions corresponding to 90% transmittance.

based on the effective concentration of ciprofloxacin moieties. As tabulated in Table 2, all samples exhibited antibacterial Table 2. MICs of Cipro-PNIPAMn against E. coli below and above LCSTCipro‑PNIPAMn MICs (μg/mL)a,b against E. coli K12 (wild type) conjugate CiproPNIPAM23 CiproPNIPAM43 CiproPNIPAM72 CiproPNIPAM90

25 °C 33 °C

on−off ratioc

MICs (μg/mL)a,b against OmpF-deficient E. coli mutant 25 °C 33 °C

Scheme 1. Schematic Illustration of How TemperatureInduced Shape-Shifting of the PNIPAM “Tail” Influenced Intracellular Access and Hence Bactericidal Activity of the Ciprofloxacin “Head” for Gram-Negative Bacteria

on−off ratioc

6

96

16

48

96

2

3

12

4

12

24

2

1.5

3

2

6

6

1

1.5

3

2

3

3

1

a

Determined by using the 2-fold broth microdilution method as per the Clinical and Laboratory Standards Institute (CLSI) guidelines. Each compound was tested in triplicate. bReported values were based on the effective concentration of ciprofloxacin moieties. cThe on−off ratio was defined as the ratio of MIC at 33 °C to that at 25 °C. An on−off ratio larger than 2 was considered significant, or switchable.

activity against E. coli K12 at 25 °C, albeit at varying potency. The biological activity should arise from the ciprofloxacin “head” alone, because the PNIPAM “tail” before conjugation exhibited no measurable inhibitory effect on growth of the strain even at a high concentration (MICs > 9000 μg/mL). In particular, the potency of the conjugate was found proportional to the length of the PNIPAM “tail”, with the MIC decreasing from 6 to 1.5 μg/mL as nNIPAM increased from 23 up to 90. Precise reason for this observation was still open to debate, but one possibility was that longer PNIPAM “tail” possessed more nonpolar isopropyls, and thus the conjugate preferred to be internalized via partitioning into the glycerophospholipid bilayer patches scattering in the OM, rather than take the porin pathway. Plausibility of this assumption was verified herein by experimentally measuring the MICs of the conjugates against one E. coli mutant lacking OmpF protein. Comparing susceptibility of the OmpF-deficient mutant with that of the wild-type strain at 25 °C revealed that the former became less vulnerable to all conjugates, and the MIC increased by different degrees for different lengths of the PNIPAM “tail” (Table 2). In the case of Cipro-PNIPAM23, the MIC against the mutant was

Previous high-resolution structure analysis revealed that OmpF had a pore size as small as 1.2 nm.27 When the “tail” in Cipro-PNIPAM23 fully extended, displaying a small crosssection perpendicular to its length, the drug “head” could still squeeze through the lumen of the OmpF channel, followed by threading of the flexible “tail”, to approach intracellular targets. The translocation was likely to proceed in such a “head-to-tail” fashion, because the ciprofloxacin “head” was rigid so that its passage would not be hindered by an entropic barrier.28 Anyway, this speculation seemed to contradict the acknowledged understanding that the upper molecular weight limit of solutes able to pass through the OmpF channels fell into a range of 500−700. To rationalize this contradiction, we should note that the solute exclusion experiment leading to this understanding was based on transport of saccharides,29,30 which exhibited quite different conformation in aqueous solution from that of Cipro-PNIPAM23 at 25 °C. Indeed, transport through porin channels is considered a complex function of physicochemical properties (e.g., molecular weight, conformaC

DOI: 10.1021/acs.bioconjchem.7b00599 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry tion, hydrophilicity, and charge) of the translocating molecules.31 Until now, many studies have reported that OmpF permitted transport of ciprofloxacin tailed with various hydrophilic, thread-like nonelectrolytes (e.g., polyethylene glycol, poly(2-methyloxazoline), and poly(2-ethyloxazoline)) that possessed a molecular weight ranging from 1382 to more than 3000.31−33 These observations, including the one herein, all seemed in contradiction with the established exclusion limit of OmpF, but reasonable, because these nonelectrolyte “tails” differed from saccharides in conformation. However, when the “tail” in Cipro-PNIPAM23 deformed into a bulky globule at 33 °C, the conjugate would get stuck at the narrow opening of the porins, excluding the drug “head” from accessing intracellular targets. Also, it was noteworthy that the PNIPAM “tail” from RAFT polymerization displayed a narrow polydispersity. This ensured that almost all Cipro-PNIPAM23 conjugates above LCST shared a commodity in geometry, and thus could be prevented from accessing the porin channels. However, hydrophobic uptake was still possible when the PNIPAM “tail” collapsed; otherwise, Cipro-PNIPAM23 should exhibit a higher on−off ratio in response to temperature. A solution to address this challenge will be reported in a follow-up paper. Furthermore, despite a switchable activity, Cipro-PNIPAM23 at 25 °C displayed compromised antibacterial potency relative to free ciprofloxacin (measured MIC = 0.1 μg/mL). To the best of our knowledge, this is a dilemma confronting all researchers in this field. For example, Ben and co-workers8 found that conjugation of a photoisomerizable spiropyran to ciprofloxacin resulted in a 2-fold difference in activity against E. coli as the spiropyran moiety isomerized upon light irradiation. However, the MIC of the conjugate in the “on” state was 50 times higher than that of free ciprofloxacin. Similarly, Anne and co-workers34 also found that the potency of a photoswitchable antimicrobial peptide incorporated with a photoisomerizable diarylethene scaffold decreased significantly compared with the parent peptide. Therefore, achieving switchable control over antibiotic activity without sacrificing the potency of the antibiotic itself is challenging, which deserves more design efforts in this field. To support the above-mentioned molecular mechanism, a set of experiments were devised to investigate how temperatureinduced “tail” shape-shifting influenced cell uptake of the antibiotic “head”. First, Cipro-PNIPAM23 was subject to dynamic and static light scattering analysis, which measured hydrodynamic radius (⟨Rh⟩) and z-average root-mean-square radius of gyration (⟨Rg⟩) of the conjugate, respectively. In Figure 2a, both ⟨Rh⟩ and ⟨Rg⟩ dropped sharply around the LCST of Cipro-PNIAPM23, reflecting temperature-induced shape-shifting of the “tail”. The inset in Figure 2a showed the temperature dependence of ⟨Rg⟩/⟨Rh⟩ ratio of the conjugate. It is known that for a random coil chain in good solvent, ⟨Rg⟩/ ⟨Rh⟩ ∼ 1.5, while for a uniform hard sphere, ⟨Rg⟩/⟨Rh⟩ ∼ 0.774.35 The experimental ⟨Rg⟩/⟨Rh⟩ ratio approaching 1.5 at 25 °C confirmed fully extended conformation of CiproPNIPAM23. Upon heating above the LCST, the ⟨Rg⟩/⟨Rh⟩ ratio of Cipro-PNIAPM23 was found always higher than that of a uniform hard sphere. According to the definitions of ⟨Rg⟩ and ⟨Rh⟩, a hard sphere with a denser shell has a higher ⟨Rg⟩ than a uniform sphere of the same size, but the density distribution has no influence on ⟨Rh⟩. Thus, a higher ⟨Rg⟩/⟨Rh⟩ than 0.774 suggested that the Cipro-PNIPAM23 globule possessed a denser shell, presumably constituted by the ciprofloxacin segments. This finding could be explained by the fact that the PNIPAM “tail” was simply too short to wrap the bulky antibiotic “head”.

Figure 2. (a) Temperature dependence of the average hydrodynamic radius, ⟨Rh⟩, and the z-average root-mean-square radius of gyration, ⟨Rg⟩, of Cipro-PNIPAM23 in water, diluted to a concentration of 100 μg/mL for ⟨Rh⟩, and a concentration of 0.5 μg/mL for ⟨Rg⟩ measurement, respectively. The inset in (a) illustrates the temperature dependence of the ratio of ⟨Rg⟩/⟨Rh⟩. (b) Fluorescence images of biotin-tagged Cipro-PNIPAM23 deposited on microscope glass slides at 25 and 33 °C, respectively, followed by incubation with streptavidinAlexa Fluor 488 assembly and rinsing at the same temperature with the deposition process. To avoid different imaging histories, the sample was imaged simultaneously with the glass substrate providing the background signal. (c) High-resolution F 1s XPS spectra of CiproPNIPAM23 deposited at 25 and 33 °C, respectively.

Or alternatively, the antibiotic moieties with hydrophilic substitutes enthalpically preferred to remain at the outermost layer of the collapsed globule, minimizing the total free energy of the system. As the “tail” deformed with increasing temperature, radial distribution of the antibiotic “head” was further confirmed by using a tagged conjugate, which was prepared by chemoselectively biotinylating the 3-carboxyl in ciprofloxacin moiety (SI, Scheme S1). The biotin moiety is stable and small so that it rarely interferes with intrinsic function of the labeled substrate.36 The tagged conjugate was then deposited at 25 and 33 °C, respectively, followed by selectively in situ staining those still-exposed antibiotic “heads” with fluorophore-labeled streptavidin at the same temperature with the deposition process. If the biotin-tagged antibiotic “heads” were buried by the PNIPAM “tail” at high temperature, streptavidin that contains multiple binding sites of high affinity for biotin would be washed away after staining. Thus, the sample deposited at high temperature should fluoresced with much lower intensity than that deposited at low temperature. Figure 2b illustrated that fluorescent streptavidin could be detected for both deposition temperatures with similar intensity, indicating that the antibiotic “heads” were still well exposed to the environment, rather than being buried within the collapsed globule upon heating. Given a signature fluorine substituent in ciprofloxacin, X-ray photoelectron spectroscopy (XPS) was also employed to investigate the change in fluorine content of Cipro-PNIPAM23, deposited at 25 and 33 °C, respectively. In accordance with the results from fluorescence labeling, the XPS signal of fluorine element was detectable for both deposition temperatures with indiscernible change in intensity (Figure 2c), indicating again that the ciprofloxacin moieties were not buried as the PNIPAM “tail” deformed. The biotinylated Cipro-PNIPAM23 was then employed to incubate with E. coli K12 at 25 and 33 °C, respectively. Following fixation and permeabilization, cellular uptake and localization of the conjugate were visualized by staining with fluorophore-labeled streptavidin. The cell nucleoids were counterstained by 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) with blue florescence as control. As shown in D

DOI: 10.1021/acs.bioconjchem.7b00599 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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membrane and cross by diffusion. This was confirmed herein by using coarse-grained (CG) molecular dynamics, in which partitioning of a Cipro-PNIPAM23 conjugate into an E. coli cytoplasmic membrane representative (a mixed phospholipid bilayer model composed of 1-palmitoyl-2-oleoyl-sn-glycero-3phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-snglycero-3-phosphoglycerol (POPG) in a proportion of 3:1) at 25 °C was observed (Figure 4).

Figure 3, temperature imposed no appreciable influence upon the uptake of biotin-tagged free ciprofloxacin, which accumu-

Figure 4. Snapshots of the partitioning dynamics of a CiproPNIPAM23 CG model into a mixed phospholipid bilayer model composed of POPE and POPG in a proportion of 3:1 at 25 °C. The beads constituting the bilayer are rendered as solid spheres, with the connectors as solid cylinders. The beads and connectors representing the conjugate are shown as dots and lines, respectively. For clarity, the CG water beads are not displayed. A video assembled by stepping through the dynamics trajectory frame sequence can be found in the Supporting Information.

Figure 3. Intracellular uptake and localization of biotinylated CiproPNIPAM23 in E. coli K12 by fluorescence microscopy and flow cytometry. Following incubation with the conjugate at different temperatures, the cells were fixed, permeabilized, and stained with streptavidin-Alexa Fluor 488 assembly for analysis. The flow cytometry data are displayed in pseudocolor dot plot, in which both x and y axes are biexponentially scaled.

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lated within and on the surface of E. coli, thus fluorescing green by streptavidin-based amplification. Similarly, green fluorescence could also be observed throughout cells incubated with biotinylated Cipro-PNIPAM23 at 25 °C, indicating that ciprofloxacin with an extended PNIPAM “tail” managed to traverse the OM of E. coli. In sharp contrast, green fluorescence signal could barely be detected within cells incubated with the same conjugate at 33 °C, consistent with flow cytometry data, in which a significantly low percentage (∼0.7%) of greenpositive cells was quantitatively distinguished. This contrast underscored the essential role played by the shape of the “tail”, which, when deforming into a bulky globule, deterred the antibiotic “head” from crossing the outermost OM barrier in Gram-negative bacteria. To attain a critical concentration at the site of action, ciprofloxacin molecules having traversed the OM in Gramnegative bacteria must contend with two further, potential diffusion barriers, including the periplasm, an aqueous cellular compartment comprising 15−20% peptidoglycan of an intermittently cross-linked woven structure, and the cytoplasmic membrane, a canonical glycerophospholipid bilayer. In principle, the periplasm itself was an unlikely diffusion barrier to Cipro-PNIPAM23 with a fully solvated “tail”, since the openings in the mesh are known to be large enough for most molecules including proteins to pass through.37 Considering the hydrophilic nature of Cipro-PNIPAM23 below LCST, the cytoplasmic membrane with a hydrophobic interior should pose a formidable diffusion barrier before the conjugate finally approached its intracellular targets. Fortunately, CiproPNIPAM23 also contained multiple hydrophobic moieties, which allowed this conjugate to partition into the cytoplasmic

CONCLUSIONS In conclusion, we have demonstrated a proof-of-concept that the activity of a model antibiotic can be switched off once a short, covalently attached PNIPAM “tail” shifts geometric shape in response to temperature. The “tail”, when stretched, allows the antibiotic “head” to traverse the OM of Gram-negative bacteria for approaching intracellular targets, while excluding drug translocation upon collapsing into a bulky globule. This strategy exploits the well-known coil-to-globule transition of PNIPAM, and the size selectivity of porin channels in Gramnegative pathogen that regulates uptake of a large proportion of antibiotics, thus representing a general approach to design antibiotics with switchable activity.

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EXPERIMENTAL SECTION Materials. Acryloyl chloride (97.0%), dichloromethane (DCM, anhydrous, ≥99.8%), triethylamine (Et3N, ≥99.0%), carbon disulfide (anhydrous, ≥99.0%), methyl α-bromoisobutyrate (≥99.0%), 1-butanethiol (99.0%), 1,4-dioxane (anhydrous, 99.8%), N,N-dimethylformamide (DMF, anhydrous, 99.8%), AIBN (98.0%, recrystallized twice from ethanol prior to use), and NIPAM (≥99.0%, recrystallized twice from toluene/n-hexane (10:90 v/v) before use) monomers were purchased from Sigma-Aldrich (St. Louis, MO). Sodium hydroxide (anhydrous pellets, ≥98.0%), acetone (≥99.9%), hydrochloric acid (37%), magnesium sulfate (anhydrous, ≥99.5%), ethanol (anhydrous, ≥99.8%), and 4-(dimethylamino) pyridine (DMAP, ≥99.0%) were obtained from Kelong Chemical Engineering Co. Ltd. (Chengdu, China). Ethyl acetate (99.0%), toluene (anhydrous, 99.0%), diethyl ether E

DOI: 10.1021/acs.bioconjchem.7b00599 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry Scheme 2. Synthesis Procedure and Chemical Structure of CTA

The mixture was cooled at 0 °C by using an ice bath, and purged with dry nitrogen for 30 min before a solution of acryloyl chloride (0.66 g, 7.12 mmol) in anhydrous DCM (10 mL) was added dropwise. After stirred under nitrogen for 15 min, the mixture was allowed to warm to ambient temperature and stirred for another 1 h. The acryloylation process was monitored intermittently by thin-layer chromatography (TLC) technique using precoated 0.25 mm Silica Gel 60 F254 plates (Merck). Crude N-acryloyl ciprofloxacin monomers were obtained by repeated precipitation in n-hexane, followed by washing with ultrapure water five times. After that, the precipitate was dried, redissolved in anhydrous DCM, and refined by silica gel chromatography (DCM-methanol 2%) to obtain the final product (1.38 g, 78% yield). Chemical structure of the product was confirmed by FT-IR, 1H and 13C NMR spectroscopies, which could be found in our previous publications.25,26 Synthesis of Cipro-PNIPAMn. Cipro-PNIPAMn was synthesized by sequential solution RAFT polymerization (Scheme 3). Herein, detailed synthesis procedure for CiproPNIPAM23 was typically introduced. Into a 50 mL Schlenk flask with a magnetic bar were added NIPAM (6.3 g, 55.8 mmol), CTA (494.9 mg, 1.86 mmol), AIBN (26.3 mg, 0.16 mmol), and 1,4-dioxane (13.5 g). The reaction mixture was degassed through three cycles of freezing and thawing, and then heated up to 60 °C. After 4 h, the polymerization was quenched by rapid cooling upon immersion of the flask into iced water. The resultant PNIPAM “tail” was precipitated into cold diethyl ether, with the precipitate collected by filtration and then redissolved into 1,4-dioxane. This precipitation−filtration procedure was repeated three times. The precipitate was finally dried at room temperature under vacuum at ambient temperature overnight to afford a yellow powder (4.3 g, 63% yield). Subsequently, N-acryloyl ciprofloxacin (0.68 g, 1.77 mmol), PNIPAM “tail” prepared above (0.92 g, 0.36 mmol), and AIBN (5.8 mg, 0.035 mmol) were mixed in a 25 mL Schlenk flask containing 6.4 g DMF. The mixture was degassed through three cycles of freezing and thawing, and then stirred at 70 °C for 4 h. After that, the polymerization was quenched by immersing the flask into iced water. The mixture was then precipitated into cold water, and subject to filtration. The filtrate obtained was dried under vacuum at ambient temperature until a constant weight, followed by redissolution into DMF. This precipitation−filtration procedure was repeated three times. Finally, the yellow solid product was dissolved into distilled water, and purified by preparative high performance liquid chromatography (HPLC) on an Agilent binary pump system using a YMC-Pack Pro C18 column (150 × 20 mm; 5 μm particle size) fitted with a guard column. The sample was eluted with a mobile phase containing acetonitrile and water in a ratio of 20:80 (v/v) at a flow rate of 5.0 mL/min, and monitored at dual wavelength of 210 and 273 nm for PNIPAM “tail” and ciprofloxacin “head”, respectively. After lyophilization, a yellow powder with a yield of 52% could be obtained. The RAFT

(anhydrous, 99.0%), n-hexane (99.0%), N-hydroxysuccinimide (NHS, ≥98.0%), and 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide hydrochloride (EDC·HCl, ≥98.5%) were supplied by Alfa Aesar (Ward Hill, MA). EZ-link hydrazide-biotin, streptavidin-Alexa Fluor 488 assembly, and DAPI were acquired from Thermo Fisher Scientific, Inc. (Waltham, MA). Ciprofloxacin in white powder (≥98.0%) was kindly supplied by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Ultrapure water with a resistivity equal to 18.2 MΩ·cm at 25 °C was obtained from a Millipore Synergy water purification system. All reagents were used without further purification unless otherwise stated. Synthesis of CTA. The RAFT agent, CTA, was synthesized using a literature procedure (Scheme 2).24 Briefly, a 20 wt % aqueous solution of NaOH (18.3 g, containing 91.5 mmol NaOH) was added into a two-necked flask equipped with a reflux condenser and a nitrogen inlet, which was precharged with a solution of 1-butanethiol (91.5 mmol) in acetone (60 mL). Under a continuous flow of nitrogen, the mixture was magnetically stirred at 10 °C for 30 min, and then 100.7 mmol carbon disulfide was added via cannula over 15 min. After stirred for another 30 min, the orange solution was allowed to warm up to ambient temperature, followed by addition of 91.5 mmol methyl α-bromoisobutyrate. Subsequently, the mixture was stirred at ambient temperature for 24 h, which was then poured into 20 mL ultrapure water, and extracted by using ethyl acetate. The combined organic phases were dried over anhydrous magnesium sulfate, filtered, and then subject to rotary evaporation, resulting in an orange-colored solid with a yield of 60%. Synthesis of N-Acryloyl Ciprofloxacin. N-Acryloyl ciprofloxacin was synthesized according to our recently disclosed method (Scheme 3).25,26 In brief, ciprofloxacin (1.52 g, 4.59 mmol) and Et3N (0.71 g, 6.89 mmol) were dissolved in 30 mL anhydrous DCM in a round-bottomed flask. Scheme 3. Synthesis Procedure and Structure of CiproPNIPAMn

F

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Bioconjugate Chemistry Table 3. RAFT Polymerization Conditions for Cipro-PNIPAMn first step

second step

conjugate

[NIPAM]/[CTA]/ [AIBN]a

temperature (°C)

time (h)

yield (%)

[NACIPb]/[PNIPAM]/ [AIBN]a

temperature (°C)

time (h)

yield (%)

Cipro-PNIPAM23 Cipro-PNIPAM43 Cipro-PNIPAM72 Cipro-PNIPAM90

30/1/0.086 55/1/0.076 85/1/0.078 101/1/0.080

60 60 60 60

4.0 3.5 3.0 2.0

63 67 65 71

5/1/0.1 5/1/0.1 5/1/0.1 5/1/0.1

70 70 70 70

4.0 4.0 4.0 4.0

52 57 54 58

a

Molar ratio. bN-acryloyl ciprofloxacin.

K12 suspension with a concentration of 1 × 108 CFU/mL in PBS. The mixture was incubated at different temperatures for 2 h, followed by centrifugation and washing with PBS at 20 °C three times. Subsequently, the cells were fixed with 4% paraformaldehyde in PBS for 20 min, and then permeabilized by using 0.1% Triton X-100 in PBS for another 20 min before stained by streptavidin-Alexa Fluor 488 assembly with a concentration of 1.0 μg/mL in PBS for 90 min. After extensive washing, cell nucleoids were counterstained by DAPI with a concentration of 10 μg/mL in distilled water for 20 min as control. The cells were stored in sterile PBS at 4 °C before fluorescence microscopy imaging. Flow cytometric analyses were performed on a BD LSR II flow cytometer (Becton Dickinson and Co., Franklin Lakes, NJ), equipped with four lasers, two light scatter detectors that measured forward and side scatter, and 11 fluorescence emission analyzer. To avoid cell coincidence, the flow rate was kept at the lowest setting (data rate: 200−300 event/s). 30,000 events were recorded for each sample, with data being analyzed by using FlowJo version 10.3 (FlowJo, LLC, Ashland, OR). Molecular Dynamics Simulation Details. Computational modeling of biological systems is challenging given the multitude of spatiotemporal scales involved. Herein, CG molecular dynamics, which neglects some of the atomistic degrees of freedom, thus enabling simulation of large-scale biomolecular processes on time scales inaccessible to all-atom models, was employed to investigate whether or not CiproPNIPAM23 could partition into the cytoplasmic membrane, a canonical glycerophospholipid bilayer, of Gram-negative bacteria. The simulation was performed using Materials Studio 8.0, a commercial software package developed by Accelrys Inc. (San Diego, CA), installed on an Intel dual-processor XEON 32 bit workstation. MS Martini, a derivative from the classical MARTINI force field originally developed for lipids, was adopted to describe the interaction between the CG beads. The bonded energy terms in MS Martini are parametrized by harmonic potential, analogous to atomistic force fields. The van der Waals interactions are described using a pairwise LennardJones (LJ) 12-6 potential, while charged beads interact via a Coulombic energy function with a relative dielectric constant εrel = 15 for explicit screening. To avoid generation of undesirable noise, the nonbonded potential energy functions are cut off at a distance of 12.0 Å with smooth shifting of the interactions and forces from 0.0 to 12.0 Å for Coulombic potential, and from 9.0 to 12.0 Å for LJ potential. Due to the use of shifted potentials, the neighbor list can be updated every 10 steps using a neighbor list cut off equal to 12.0 Å. To accurately reproduce the cytoplasmic membrane of E. coli, which contains 70−80% phosphatidylethanolamine (PE) and 20−25% phosphatidylglycerol (PG),39 a mixed phospholipid bilayer CG model composed of POPE and POPG in a proportion of 3:1 was constructed. Following the procedure

polymerization conditions for all the other Cipro-PNIPAMn conjugates were summarized in Table 3. Characterization. Dynamic and static light scattering measurements were performed on a BI-200SM wide angle laser light scattering system (Brookhaven Instruments Corporation, U.S.) equipped with a MGL-III model 100 mV He−Ne laser (λ = 532 nm), a computer-controlled BI-200SM goniometer, and a BI-9000AT digital correlator. 1H and 13C NMR spectra were collected at 20 °C on a Varian 500 MHz NMR instrument in deuterated dimethyl sulfoxide (DMSO-d6, 99.9 atom % D) containing 0.03% (v/v) tetramethylsilane (TMS) as the internal reference. SEC was carried out at 20 °C on a Waters 2690D separation module equipped with a TSKGEL column and a Waters 2410 refractive index detector, where tetrahydrofuran containing 3 wt % triethylamine with a flow rate of 0.5 mL/min was employed as the eluent. Waters millennium module software was used to calculate the molecular weight based on a universal calibration curve generated by polystyrene standard with a narrow molecular weight distribution. XPS measurements were performed on an AXIS Ultra DLD instrument (Kratos, U.K.), equipped with a standard and monochromatic Al Kα X-ray excitation source (1486.6 eV). The binding energies were corrected by referencing the C 1s peak at 284.6 eV. FT-IR spectra were collected using a Nicolet iS10 FT-IR spectrometer (Thermo Scientific, U.S.) over a wavenumber range from 600 to 3000 cm−1 after 64 scans at a resolution of 2 cm−1. Turbidimetry was carried out on a UV−vis spectrophotometer (UV-3600, SHIMADZU, Japan) equipped with an external thermostat (Neslab RTE digital circulating water bath) using a quartz cuvette of 10 mm path length. The aqueous sample solutions (concentration = 1.0 g/L) were heated at a rate of 0.2 °C/min, and the transmittance at 500 nm was recorded as a function of temperature from 24 to 38 °C. Fluorescence microscopy imaging was performed on a Zeiss Axioplan2 upright microscope. Images were acquired using a Hamamatsu OrcaER cooled CCD camera with MetaMorph acquisition software when the slides were observed under a 100× oil immersion objective. E. coli K12 and one mutant lacking OmpF protein that was isolated from the wild-type strain according to a literature procedure38 were selected as indicators in antibacterial experiment. They were cultivated in Luria−Bertani broth at 37 °C for 15 h, followed by centrifugation and washing with phosphate buffer saline (PBS) three times. Before use, the bacteria were diluted to a concentration of 1 × 108 colonyforming units (CFU)/mL with PBS. MICs were measured using the 2-fold broth microdilution method according to the CLSI guidelines. To investigate the “tail” shape-dependence of cellular uptake of the antibiotic “head”, 200 μg biotin-tagged Cipro-PNIPAM23 (see SI for synthesis details) was dissolved into 2 mL E. coli G

DOI: 10.1021/acs.bioconjchem.7b00599 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry described by Marrink et al.,40 POPE, POPG, water, and CiproPNIPAM23 molecules were coarse grained by mapping approximately four heavy atoms and associated hydrogen atoms into one CG interaction bead with an average mass of 72 amu and an effective radius of 2.35 Å. Detailed mapping scheme for the above-mentioned molecules was illustrated in Scheme 4. In MS Martini potential, an equilibrium bond

algorithm was employed to main a constant pressure. The time step was set to 20 fs for all dynamics runs.

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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/acs.bioconjchem.7b00599. Details for preparation of biotin-tagged Cipro-PNIPAM23 and biotin-tagged ciprofloxacin. Experimental results: FT-IR, 1H and 13C NMR spectroscopies of CTA, 1H NMR spectroscopies of ciprofloxacin and CiproPNIPAMn, size-exclusion chromatography of PNIPAM “tail”, plot of the changes of transmittance (%) as a function of temperature for aqueous solutions of PNIPAM “tail”, 1H NMR spectroscopies of biotin-tagged Cipro-PNIPAM23 and ciprofloxacin (PDF) Video showing the partitioning dynamics of a CiproPNIPAM23CG model into a mixed phospholipid bilayer CG model composed of POPE and POPG in a proportion of 3:1 at 25 °C (ZIP)

Scheme 4. Mapping Strategies for the Chemical Structure of (a) POPE, (b) POPG, (c) Water, and (d) Cipro-PNIPAM23 Molecules in CG Dynamics Modelinga

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. a

The CG beads are shown as semi-transparent spheres, mapped over the all-atom models. For clarity, hydrogens are only shown for the atomistic water. In addition, the CG bead types assigned according to the MS Martini force field are also indicated.

ORCID

Jinming Chang: 0000-0001-5069-2824 Yi Chen: 0000-0001-6127-0248 Haojun Fan: 0000-0003-4827-3721 Author Contributions

distance of 4.7 Å and an equilibrium angle of 180° were applied for all force field types, except the glycerol ester-phosphateglycerol ester bond, which used a modified nonlinear bond angle of 120°, as well as a force constant of 10.8 kcal/mol. In the POPE representation, a positive charge was assigned to the ethanolamine group, while the phosphate groups in both POPE and POPG bore a negative charge. To build the mesomolecules into a bulk structure, a simulation cell with a dimension of 84 × 84 × 120 Å3 that contained a slab former of depth 44 Å in the center was constructed, followed by packing the POPE and POPG models into the slab in a proportion of 3:1, with the head beads sticking to the surface. This slab was then solvated with water beads, into which a Cipro-PNIPAM23 CG model was randomly inserted, yielding a simulation cell with an initial density of 1.0 g/mL. Finite-size effects were prevented by periodic boundary conditions in all three dimensions. Subsequently, optimization was carried out in two steps, by first optimizing the intermolecular interactions, followed by an optimization of all degrees of freedom. To this end, the POPE, POPG, and CiproPNIPAM23 CG models in the cell were regarded as motion groups, which were kept rigid over a minimization procedure using the Smart algorithm. After that, another geometry optimization of the same quality was performed without constraining the motion groups, but allowing the cell parameters to be optimized. The simulation cell was then subject to a long dynamics equilibration using an NPT (T = 25 °C; P = 1 bar) ensemble. Full equilibration was validated by the observation that the density of the cell fluctuated randomly about a constant mean value with increasing simulation time. Throughout the simulation, the temperature was kept constant using the Nose algorithm, while the Andersen thermostat

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support of this work by National Key Research and Development Program of China (2017YFB0308600), and Science and Technology Planning Project of Sichuan Province (2017JY0218). We also acknowledge the Analytical & Testing Center of Sichuan University for providing computational resources.

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REFERENCES

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DOI: 10.1021/acs.bioconjchem.7b00599 Bioconjugate Chem. XXXX, XXX, XXX−XXX