Surface Ligand Density of Antibiotic-Nanoparticle Conjugates

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Surface Ligand Density of Antibiotic-Nanoparticle Conjugates Enhances Target Avidity and Membrane Permeabilization of Vancomycin-Resistant Bacteria Marwa M. Hassan, Andrea Ranzoni, Wanida Phetsang, Mark A. T. Blaskovich, and Matthew A. Cooper* Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia S Supporting Information *

ABSTRACT: Many bacterial pathogens have now acquired resistance toward commonly used antibiotics, such as the glycopeptide antibiotic vancomycin. In this study, we show that immobilization of vancomycin onto a nanometer-scale solid surface with controlled local density can potentiate antibiotic action and increase target affinity of the drug. Magnetic nanoparticles were conjugated with vancomycin and used as a model system to investigate the relationship between surface density and drug potency. We showed remarkable improvement in minimum inhibitory concentration against vancomycin-resistant strains with values of 13−28 μg/mL for conjugated vancomycin compared to 250−4000 μg/mL for unconjugated vancomycin. Higher surface densities resulted in enhanced affinity toward the bacterial target compared to that of unconjugated vancomycin, as measured by a competition experiment using a surrogate ligand for bacterial Lipid II, N-Acetyl-L-Lys-D-Ala-D-Ala. High density vancomycin nanoparticles required >64 times molar excess of ligand (relative to the vancomycin surface density) to abrogate antibacterial activity compared to only 2 molar excess for unconjugated vancomycin. Further, the drug-nanoparticle conjugates caused rapid permeabilization of the bacterial cell wall within 2 h, whereas no effect was seen with unconjugated vancomycin, suggesting additional modes of action for the nanoparticle-conjugated drug. Hence, immobilization of readily available antibiotics on nanocarriers may present a general strategy for repotentiating drugs that act on bacterial membranes or membrane-bound targets but have lost effectiveness against resistant bacterial strains.

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INTRODUCTION Exposure to antibiotics induces evolutionary mechanisms that render bacteria progressively resilient to drug action.1 Recent reports indicate a continuous rise in the number of discovered and reported resistance mechanisms of superbugs to current antibiotics.2 As a result, limited options remain for treating infections caused by resistant bacteria. Methicillin-resistant S. aureus or MRSA, which is responsible for community and hospital infections, has been treated for many years with vancomycin. This glycopeptide antibiotic binds to the terminal D-alanyl-D-alanine residues of Lipid II and nascent peptidoglycan cell wall precursors, thus blocking bacteria transglycosylases and transpeptidases required for peptidoglycan biosynthesis and cross-linking. Because the bacterial cell wall of Grampositive bacteria is composed largely of peptidoglycan, vancomycin and the related glycopeptide antibiotics prevent cell division and are bacteriostatic in action. Bacterial strains have acquired partial resistance by thickening their cell wall as in the case of vancomycin-intermediate S. aureus (VISA), producing more “decoy” Lipid II and partially synthesized peptidoglycan that block glycopeptide activity by introducing an overabundance of D-alanyl-D-alanine binding sites. Alternatively, a higher level of resistance has been acquired by Published XXXX by the American Chemical Society

horizontal gene transfer of the van gene cluster from Enterococcus faecalis (VRE) to MRSA.3−5 This gene encodes for expression of D-alanyl-D-lactate instead of D-alanyl-D-alanine, removing a key ligand-target hydrogen bond interaction that results in a 1000-fold reduction in vancomycin binding affinity.6,7 Resistance spreads very rapidly as it provides an additional evolutionary advantage to a subpopulation, and it is already apparent that vancomycin may be nearing the end of its effective general use with “resistance creep” leading to increased adverse outcomes.8 Strategies to fight emerging resistance include reducing unnecessary use of antibiotics by informing the general public and physicians on the appropriate practice and developing improved rapid diagnostics, but these approaches do not help cure a resistant infection. Unfortunately, very few new drugs are presently in the antibiotic pipeline because of the technical and economic challenges to discover and develop a safe antibiotic.2,9−11 The discovery of completely novel antibiotics is rare with the most successful Received: September 4, 2016 Revised: November 10, 2016

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DOI: 10.1021/acs.bioconjchem.6b00494 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry

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Figure 1. (blue box): (I) Synthesis of azide vancomycin. (II) Conjugation chemistry of azide vancomycin to magnetic nanoparticles (NPs) with click chemistry. (red box) Bacterial membrane damage hypothesis showing the binding ratio of Lipid II terminal D-alanyl-D-alanine residues to one molecule of (A) unconjugated vancomycin, (B) low density Van-NPs, and (C) high density Van-NPs. The figure illustrates the reason behind the difference in binding potency of 1 molecule of unconjugated vancomycin compared to Van-NPs at different local densities.

polymeric hydrogel, resulting in enhanced potency.17 This study hypothesized that the enhanced potency was a consequence of the increased local density of vancomycin and wide contact area with the bacterial cell wall.17 Naoto et al. prepared vancomycin-dextran nanocolloid nanoparticles (NPs) and tested their effect on the cellular morphology of VSE and VRE strains. Although their conjugated vancomycin quantification was indirect (based on spectrophotometry), they showed high antibacterial potency against vancomycin-resistant enterococci with vancomycin-NPs (minimum inhibitory concentration (MIC): 8 μg/mL) compared to vancomycin alone (MIC: >256 μg/mL).18 Other studies showed variable bacterial inhibition profiles for NP-conjugated vancomycin when functionalized in different conformations.19−21 We now describe a quantitative method to more accurately tailor the antibacterial potency of vancomycin conjugated on nanocarriers and study its biological effect. We have demonstrated controlled functionalization of the nanocarriers with different vancomycin local densities and proved that higher local density leads to superior antibacterial activity, bacterial affinity, and increased membrane damage. This study demonstrates the relationship between unconjugated antibiotic

strategy to develop new antibiotics based on rational structural modification of already approved drugs in an attempt to improve potency and overcome resistance. For glycopeptide antibiotics, recently approved dalbavancin and oritavancin introduced semisynthetic modifications that greatly improved potency and provided an extended half-life.12 Yarlagadda et al. synthesized vancomycin carbohydrate conjugates that showed higher bactericidal activity and binding affinity against VRE strains,13 and Boger et al. created new vancomycins with an amidine designed to re-establish the hydrogen bond lost in VanA mutants.14 Recently, Yarlagadda et al. modified vancomycin with a dipicolyl substituent to bind bacterial Zn2+ and complex with cell-wall pyrophosphates. These dipicolylvancomycin derivatives showed 375-fold enhancements in activity against VRE strains.15 A different approach was employed by Lei et al. who conjugated penicillin-G to silica nanoparticles and tested the variation in inhibitory effect when immobilized on a nanocarrier. Their work demonstrated that drug immobilization on nanoparticles translated in slightly larger inhibition zones when tested using an antibacterial disc diffusion method.16 These findings were further supported by a subsequent study where vancomycin was immobilized onto a B



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(N3-vancomycin; Figure 1, compound 2) was synthesized by amide coupling between azido-PEG3-amine and the vancomycin C-terminal carboxyl group, reacting vancomycin hydrochloride with azido-PEG3-amine in the presence of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and N,N-diisopropylethylamine (DIPEA) in DMF (Figure 1I). The adduct was characterized by NMR and LCMS (Figures S2 and S3). We tested the antibacterial potency of the azide-modified vancomycin by testing its MIC against Gram-positive bacteria (see Table 2). The nanoparticle component was constructed with a three step surface molecular architecture. A monolayer of human serum albumin (HSA) was bound onto the surface of commercially available superparamagnetic carboxylated 170 nm nanoparticles by carbodiimide carboxyl activation to mitigate nonspecific interactions when deployed in biological fluids. HSA molecules were then used to anchor an N-hydroxy succinimide (NHS)-activated dibenzocyclooctyl (DBCO) hydrophilic PEG linker. The strained DBCO alkyne was subsequently reacted with the N3-vancomycin component via a biorthogonal Cu-free “click” [3 + 2] azide−alkyne cycloaddition reaction25 to generate a triazole linkage, resulting in covalently vancomycin-functionalized NPs (Van-NPs) (Figure 1II). Characterization of Van-NPs. Each step of the surface molecular architecture was carefully characterized to ensure consistent covalent binding of N3-vancomycin to the NP surface. The generation of an HSA layer was assessed by bicinchoninic acid assay (BCA) that was quantified with a dilution series of a standard HSA protein (∼2 × 106 HSA molecules/μm2). Transmission electron microscopy imaging of HSA-NPs showed HSA protein corona (Figure S4). Introduction of the DBCO layer was confirmed by binding a fluorescent dye with complementary azide functional moiety (N3-NBD); comparison with a reference curve enabled quantification of conjugated DBCO (∼4 × 104 DBCO molecules/μm2) (Figure 2A). Once vancomycin was “clicked” onto the nanoparticle surface, we validated the coupling efficiency indirectly by quantifying the amount of N3-NBD binding to unreacted DBCO molecules. Additionally, we quantified functionally active conjugated vancomycin using two methods: (1) a fluorescent measurement of a fluorophore (carboxyfluorescein, Fam) attached to a vancomycin-binding ligand, Fam-Lys-D-Ala-D-Ala (Fam-Kaa) and (2) LCMS analysis of eluted Ac-Kaa after being incubated with the vancomycin nanoparticles (Van-NPs). N-Acetyl-L-Lys-D-Ala-D-Ala (Ac-Kaa) (Figure S5A) is a synthetic vancomycin target ligand that mimics the peptidic component of Lipid II involved in glycopeptide binding. Ligand-based quantification methods were developed to assess functionally active vancomycin molecules only rather than total conjugated vancomycin. For the fluorescent indirect quantification of vancomycin coupling efficiency, we incubated a constant amount from all prepared Van-NP batches with N3-NBD to quantify the amount of vancomycin-coupled DBCO molecules with subtraction from the initial measurement of unreacted DBCO molecules providing an estimation of the amount of DBCO molecules/ μm2. For the fluorescent Fam-Kaa direct determination, similarly, a constant amount from all Van-NP batches was incubated with Fam-Kaa. Unbound dye was removed by multiple washing steps, and then comparison of the fluorescence intensity of the washed nanoparticles with a reference curve enabled estimation of the surface density of

or conjugated antibiotic local density and the resulting antibiotic potency and binding affinity.

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RESULTS AND DISCUSSION Synthesis of Azide-Vancomycin and Conjugation of Van-NPs. An azide-modified vancomycin precursor with a polyethylene glycol (PEG) linker was synthesized and then coupled to the nanoparticle surface by strain-promoted cycloaddition with a cyclooctyne substituent that was attached to the nanoparticle by another PEG linker (Figure 1). Van-NPs were prepared with a wide range of vancomycin input concentrations, and the resulting functionalized particles were categorized under three different vancomycin local densities: high, intermediate, and low density. We define local density as the number of functionally active molecules per unit surface area (vancomycin molecules/μm2) (surface area per bead is ∼0.1 μm2, assuming that the beads are spherical with negligible surface roughness) (Table 1). Local density cutoffs were identified based on the particles’ biological differences and only used throughout the text for ease of discussion. Table 1. Quantified Values of Van-NPs (Conjugated Vancomycin) Based on the Fluorescence Signal of Bound Fam-Kaaa local density classification low density

intermediate density high density

Van-NP batch number

vancomycin concn of 1 mg of Van-NPs (based on Fam-Kaa) (μg/mL) ± ± ± ±

1 2 3 4

0.200 2.00 3.65 4.33

0.016 0.264 0.098 0.194

5 6

8.74 ± 0.083 11.75 ± 0.096

a

Definition and calculations of local densities are based on the estimated number of particles: 1.76 × 1013 particles/mL. Different Van-NP batches were prepared with increasing vancomycin concentration and categorized into corresponding local densities based on the batches’ biological data. Data (n = 3) are shown as means ± SD.

All prepared Van-NP batches with different vancomycin concentrations were carefully quantified and tested for biological activity. When tested for their antibacterial properties, we found that intermediate and high local density Van-NPs resulted in 19- to >100-times lower MIC against VISA and VRE strains, respectively, and also showed higher binding avidity and bacterial membrane permeability. We hypothesize that intermediate and high density Van-NPs allow for multiple more strongly localized binding events (more than a ratio of 1:1) on the bacterial cell wall, whereas when the unconjugated drug is in solution, Brownian diffusion randomizes the binding events over a wider surface area. Accordingly, a high drug density on nanocarriers caused rapid and localized membrane damage (Figure 1). The ligand-binding region of vancomycin resides in the heptapeptide backbone; therefore, a number of regions outside of this area can be derivatized to facilitate coupling onto the nanoparticles without impairing potency. Sites with accessible functional groups include the C-terminal carboxy group vancosamine or N-terminal Leu primary and secondary amine groups, and hydroxyl and phenolic groups. All of these sites have been previously used to generate vancomycin derivatives (Figure S1).22−24 Our azide-modified vancomycin derivative C



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Table 2. MIC Values Comparison of Compounds against Vancomycin Sensitive and Resistant Strainsa fold improvement in MIC to resistance profile

bacterial strain

sensitive resistant

MRSA MRSA VRSA (VanA) VRSA (VanA) VRE (VanA) VRE (VanB)

sensitive

S. aureus (ATCC 25923) S. aureus (ATCC 43300) S. aureus (ATCC 33591) S. aureus (NARSA VRS1) S. aureus (NARSA VRS4) E. faecium (ATCC 51559) E. faecalis (ATCC 51299) Escherichia coli (ATCC 25922) Klebsiella pneumoniae (ATCC 13883)

vancomycin MIC (μg/mL)

N3-vancomycin MIC (μg/mL)

N3vancomycin

Van-NPs MIC (μg/mL)

vancomycin

1

7.8

0.52 ± 0.04 (low density)

15

2

1

8


10

1

1

8


10

1

64

250

13.3 ± 1.32 (intermediate density)

19

5

>64

>128

10.02 ± 0.87 (intermediate density)

>13

>6

512

4000

28.9 ± 0.94 (high density)

>100

18

512

4000

28.9 ± 0.94 (high density)

>100

18

>64

>64

>50 (high density)

not applicable

>64

>64

>50 (high density)

not applicable

a

MIC value reflects the calculated conjugated vancomycin content based on vancomycin density and number of nanoparticles. Data (n = 3) are shown as means ± SD. The MIC experiment was repeated twice.

Figure 2. (A) Quantification of DBCO layer formation as measured by the fluorescence signal of N3-NBD dye. Data (n = 3) are shown as means ± SD; error bars are too small to be visible in the graph. (B) Comparison between the three quantification methods of conjugated N3-vancomycin based on binding of Fam-Kaa, calculated reacted cyclooctyne detected by N3-NBD fluorescence, or binding then elution of Ac-Kaa measured by LCMS. Data (n = 2) are shown as means ± SD; most error bars are too small to be visible in the graph.

mean particle size (Figure S7). We postulate that both of these observations may be due to the tendency of vancomycin to form intra- and internanoparticle dimers at high concentrations (∼745 mol/dm3).26,27 Determination of MIC. To create Van-NPs with varying local densities, we varied the vancomycin concentration in different NP batches while keeping constant the amount of NPs (Table 1). We prepared different batches of Van-NPs (1.76 × 1013 particles/mL) with a 2-fold serial dilution of added vancomycin and tested their MIC against vancomycin-sensitive and -resistant strains (the Van-NPs vancomycin content for MIC determinations was calculated based on the Fam-Kaa assay and varied from 0.2 to 30 μg/mL). HSA-NPs were used in MIC studies as a positive control to confirm that the coated NPs themselves did not possess any antimicrobial activity. We found that at intermediate (batch 4) and high Van-NP density (batch 6), inhibition of bacterial growth occurred against VRSA and VRE strains, whereas inhibition of sensitive and MRSA strains occurred with low density Van-NPs (batches 1 and 3),

active vancomycin covalently bound onto the NPs. For these results to be confirmed, the prepared Van-NP batches were also incubated with an excess concentration of Ac-Kaa; after washing away unbound Ac-Kaa, the remaining Ac-Kaa bound to vancomycin was eluted using 100 mM HCl with the concentration of the Ac-Kaa in the eluent measured by LCMS (Figure S6). Our results showed comparable functionally active vancomycin local density quantification by all three methods (Figure 2B and Table S1) though the Fam-Kaa and N3-NBD fluorescence-based assays were more sensitive, requiring 50and 10-times less NPs than the LCMS method, respectively. For further discussion in this paper, we have relied on the local density based on the Fam-Kaa measurement (see Table 1). During our experiments, we observed reversible nanoparticle aggregation at high vancomycin loading concentrations with aggregation easily dissipated by exposure to ultrasound. We used dynamic light scattering (DLS) to monitor the evolution of the hydrodynamic volume for progressively increasing vancomycin loadings and found a corresponding increase in D



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respectively. Turbidity was observed with HSA-NPs due to microbial growth. Notably, we observed 15- to >100- and 2- to 19-fold improvements in MIC with the Van-NPs compared to free N3-vancomycin and unmodified vancomycin, respectively, when tested against sensitive and vancomycin-resistant S. aureus and VanA and VanB resistant Enterococcus strains (Table 2). MRSA strains showed 1- to 10-fold enhancements in MIC compared to unmodified vancomycin and N3-vancomycin (Table 2), respectively. Vancomycin, N3-vancomycin, and high density Van-NPs showed no inhibitory activity against sensitive Gram-negative bacteria (Table 2). This significant enhancement of vancomycin potency showed that bacterial inhibition was affected by the local drug density with even greater improvements in activity compared to that of free vancomycin against the VISA and VRE strains. We progressively titrated the NP concentration (200 to 5 mg/mL) for a given local density of vancomycin and investigated the concentration of nanoparticles required to achieve inhibition of bacterial growth for a given local density of vancomycin. The results showed an indirect relationship between vancomycin local density and the number of NPs required for inhibition: higher densities required fewer nanoparticles for both vancomycin-sensitive and -resistant strains (Figure S8A). When MIC values for each strain were calculated based on the conjugated vancomycin content (based on the vancomycin loading and the number of nanoparticles required for inhibition), the MICs were found to increase with increasing local density (Figure S8B). For the vancomycinsensitive S. aureus strain (ATCC 25923), an MIC of 0.52 μg/ mL was detected with a low density (2.18 × 102 vancomycin/ μm2) but high number (25 mg/mL of NPs) of Van-NPs. Increasing the local density to 2.41 × 103 or 3.94 × 103 vancomycin/μm2 required a reduced number of NPs (15 and 12.5 mg/mL, respectively), resulting in MIC values corresponding to 3.3 and 4.5 μg/mL of vancomycin, respectively. For a resistant S. aureus strain (NARSA VRS-1), an MIC of 13.3 μg/ mL of vancomycin was obtained with intermediate density VanNPs (6.28 × 103 vancomycin/μm2 at 25 mg/mL of NPs), whereas a higher MIC of 15.2 μg/mL was obtained with a higher local density (9.56 × 103 vancomycin/μm2 at 17.5 mg/ mL of NPs). Low density Van-NPs were unable inhibit the growth of the resistant strain even with a very high number of NPs. The results demonstrate that the MIC value is a result of both the local density of vancomycin on the surface of the nanoparticles and the number of nanoparticles, which together represents the number of binding events occurring on the bacterial membrane for efficient killing. Depending on the resistance profile of the strain, a specific local density is required: higher local density is required with highly resistant strains than with sensitive strains. However, for a given strain, the lowest possible local density gives the lowest MIC value (based on calculated conjugated vancomycin content). Vancomycin Binding Affinity and Membrane Permeability. To elucidate the fundamental mechanism behind enhanced drug potency when localized on nanocarriers, we tested the binding affinity of vancomycin, N3-vancomycin, and Van-NPs to the bacterial ligand target by using a ligand displacement assay to assess what concentration of competing Ac-Kaa ligand was required to abrogate antibacterial activity. The test compounds were assessed at 10-fold MIC in the presence of a wide range of Ac-Kaa molar excess. The displacement by Ac-Kaa is used to demonstrate the differences in affinities to the native bacterial Kaa target of Lipid II.28,29

The design of the experiment is based on calculating the AcKaa concentration required for the vancomycin molecules to favor binding to Ac-Kaa instead of inhibiting bacterial growth: higher Ac-Kaa concentration requires higher vancomycin bacterial binding affinity. The results showed that vancomycin, N3-vancomycin, and low density Van-NPs (batch 3) had the same affinity to bacterial ligand with >2-fold molar excess of AcKaa (based on vancomycin molecular weight) resulting in bacterial growth (Figure 3A). However, intermediate (batch 4) and high density (batch 6) Van-NPs showed a stronger affinity to bind to bacteria, requiring >4 and 64 molar excess of Ac-Kaa, respectively, to overcome bacterial inhibition (Figure 3A). Our findings demonstrate that there is a direct relationship between increasing the local density of drug-conjugated nanocarriers and bacterial binding affinity. To determine the extent of bacterial cell wall damage, we incubated S. aureus with vancomycin (at 20-fold MIC) and Van-NPs of different local densities (at 10-fold MIC) in the presence of propidium iodide and measured the fluorescence over time. Controls of S. aureus with phosphate buffer and HSA-NPs were incubated for the same time in the presence of propidium iodide. Propidium iodide (PI) is a fluorescent dye that assesses bacterial viability and membrane integrity through increase of its fluorescence when bound to the bacterial nucleic acid content.30,31 Fluorescence signal values of vancomycin and Van-NPs were subtracted from controls of phosphate buffer and HSA-NPs, respectively. Remarkably, vancomycin did not show any membrane damage after 10 h, whereas all densities of Van-NPs showed membrane permeabilization of PI after 1 h of incubation (Figure 3B). The high density Van-NPs (batch 6) showed higher fluorescence signal (2800) compared to low (batch 3) and intermediate density (batch 4) Van-NPs (2200), indicating that higher localized drug concentration induced considerably more membrane leakage. Surprisingly, the low density Van-NPs triggered earlier membrane damage (increased fluorescence seen at the first 5 min compared to 20−25 min with other Van-NP densities), which may be due to the higher number of NPs (NP concn: 25 mg/mL) compared to intermediate and high density Van-NPs (NP concn: 10 mg/ mL) leading to increased binding events and binding kinetics rate. To confirm the Van-NPs’ effect on the bacterial membrane integrity, we did membrane depolarization experiments using 3,3′-dipropylthiadicarbocyanine iodide (DiSC 3 (5)) dye. DiSC3(5) is a cationic dye that accumulates in hyperpolarized cell-membranes and shows fluorescence self-quenching.32−35 Bacterial membrane permeabilization causes depolarization of the membrane and results in the release of the dye, increasing its fluorescence signal. S. aureus strain was incubated with DiSC3(5) dye and monitored for fluorescence self-quenching for 1 h (Figure 3D); then, vancomycin, N3-vancomycin, and Van-NPs of different local densities (at their MIC concentration) were added, and the fluorescence signal was monitored over time. Controls of S. aureus with sterile water, 0.1% TritonX, and HSA-NPs were incubated under the same conditions. Fluorescence values of vancomycin and Van-NPs were normalized against controls. The fluorescence signal values showed that vancomycin and N3-vancomycin (700) showed no membrane depolarization, whereas all densities of Van-NPs showed high levels of membrane permeabilization (4000− 15000) (Figure 3C). Low density Van-NPs showed the lowest fluorescence signal (4000), whereas intermediate (14000) and high density (15000) showed the maximum membrane E



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membrane was visualized with transmission electron microscopy. High density Van-NPs at vancomycin concentrations below MIC (0.05 μg/mL) were incubated with sensitive S. aureus strain for 4 h; then, the samples were prepared for imaging. Figure 3E shows bacterial membrane rupture at the Van-NP binding sites even at such low vancomycin concentration. To determine bacterial ATP leakage, we incubated the S. aureus bacterial strain with different densities of Van-NPs and vancomycin at their MIC concentration for 1.5 h before the ATP concentration was determined in the samples’ supernatants. Vancomycin was used as a control because previous studies showed that it does not cause any leakage to cellular ATP36 and to compare the effect of nanoconjugation along with different densities. Low density Van-NPs and vancomycin showed comparable total ATP concentration (Figure 3F). Intermediate and high density Van-NPs showed slightly higher total ATP concentration (0.3−0.4 nM) (Figure 3F), which confirms that controlled vancomycin density had potentiated vancomycin’s effect and caused slight leakage of cellular ATP. In this study, the binding affinity and membrane damage results’ supported the membrane damage hypothesis of high density Van-NPs, where multiple vancomycin molecules cause higher binding avidity that lead to localized and severe membrane damage in a concentrated surface area at the same time. This increased membrane damage of high density VanNPs increased the Van-NPs affinity against resistant bacteria with thickened cell walls and compensated for the modification of the Lipid II target ligand in the VanA and VanB resistant strains.

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CONCLUSIONS In summary, the controlled conjugation of antibiotics onto nanocarriers resulted in enhanced antibiotic efficacy with improved MIC (based on conjugated antibiotic content) against both sensitive and resistant strains with optimum local density depending on the bacterial strain. The conjugated nanoparticle antibiotic showed increased affinity to the bacterial binding targets, and the high local density induced significant bacterial membrane damage in 2 h compared to no damage by the free antibiotic after 10 h. As a consequence, there is a new hope to target superbugs using current antibiotics by designing nanotechnologies to immobilize readily available antibiotics on biocompatible nanocarriers.37 Through the use of recent advances in site-directed biodegradable NPs for therapeutic applications,38−40 different polymers of proven stability may be used to functionalize current antibiotics and renew their potentiated activity against resistant strains. Our results suggested that conjugation of antibiotics on nanocarriers may have different mechanisms for interaction with bacteria that requires further investigation in the future. The finding is of potential impact to tackle drug resistance and may prevent the development of new resistance mechanisms.

Figure 3. Comparison of bacterial inhibition by unconjugated vancomycin and Van-NPs. (A) Binding affinity of unconjugated and conjugated vancomycin and bacterial ligand as measured by prevention of its inhibitory activity in the presence of added synthetic Ac-Kaa. Data (n = 3) are shown as means ± SD. (B) Membrane permeability of propidium iodide to bacteria in the presence of vancomycin and Van-NPs with different loading densities. (C) Membrane permeability using DiSC3(5) dye in the presence of vancomycin, N3-vancomycin, and Van-NPs with different local densities (1× MIC). (B,C) Fluorescent values of Van-NPs and vancomycin were subtracted from the average fluorescent values of HSA-NPs and negative controls incubated under the same conditions, respectively. (D) DiSC3(5) fluorescence self-quenching over time (min) compared to bacterial cells only. (E) Transmission electron microscopy imaging (JEOL 1011) of ruptured bacterial membrane after treatment with high density Van-NPs (0.05 μg/mL, below MIC) (arrows) (scale bar = 1 μm). (F) Total cellular ATP leakage concentration in the presence of vancomycin and Van-NPs with different densities (1× MIC). All data (n = 2) are shown as means ± SD; some error bars are too small to be visible in the graphs. All experiments were repeated twice.

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EXPERIMENTAL PROCEDURES Synthesis of N3-Vancomycin. Vancomycin hydrochloride (567 mg, 3.82 × 10−4 mol), PyBOP (219 mg, 4.20 × 10−4 mol), and azido-PEG3-amine (N3(CH2CH2O)3CH2CH2NH2, 100 mg, 4.58 × 10−4 mol) in DMF (30 mL) were stirred until dissolved. DIPEA (532 μL, 3.05 × 10−3 mol) was added slowly to the reaction mixture. The reaction was stirred at room temperature for 2 h, followed by removal of solvent under

permeabilization at the MIC concentration (Figure 3C). We noticed that the control HSA-NPs showed membrane depolarization (Figure S9), but interestingly, the conjugation of vancomycin potentiated the membrane permeabilization of the drug significantly. Also, the effect of Van-NPs on bacterial F



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mM HCL and incubated for 15 min at room temperature. Then, the supernatant was diluted 1:1 with PBSP and measured on LCMS. Determination of MIC Using Serial Dilution Method. Determination of MIC was done using the broth serial dilution method according to National Committee for Clinical Laboratory Standards (NCCLS). Sensitive and resistant S. aureus strains were cultured on Muller Hinton broth (MHB) (Bacto Laboratories, Cat. No. 211443) at 37 °C overnight. Then, the bacterial strains were subcultured and incubated at 37 °C until they reached mid log phase when they were diluted to a final MIC inoculum concentration of 5 × 105 cfu/mL in 100 μL as a final volume. For vancomycin and N3-vancomycin, MICs were determined by a 2-fold serial dilution of 50 μL of 128 and 1024 μg/mL, respectively, of the drugs in 96-well nonbinding surface plates (NBS, Corning). For Van-NPs, different local densities of Van-NPs (final vancomycin input concn range = 0.125−3 mg/mL) and different NPs concentrations of each local density (final NPs concn = 25 to 5 mg/mL) were used in a final volume of 50 μL (washed 3 times with Muller Hinton broth) in sterile eppendorfs for MIC determination. HSA-NPs were used as a positive control. All eppendorfs and 96-well plates were kept at 37 °C under rotation for 24 h. MICs were the lowest concentration that showed no visible growth. Vancomycin Binding Affinity Assay. Binding affinity was determined by incubating the same prepared MIC bacterial inoculum with vancomycin, N3-vancomycin, and Van-NPs (concn: 10× MIC) with increasing molar excess of Ac-Kaa (WUXI ZHONGKUN Biochemical Technology) in Muller Hinton broth (MHB) at 37 °C for 24 h under rotation. Vancomycin, N3-vancomycin, and Van-NPs binding affinity were determined by the highest concentration of Ac-Kaa required that still allowed bacteria killing, hence, showed no visible growth. Vancomycin Membrane Permeabilization Assay. Bacterial membrane permeabilization was determined by incubating sensitive S. aureus strain ATCC 25923 with vancomycin (concn: 20× MIC) and Van-NPs (concn: 10× MIC) (in PBSP) in the presence of propidium iodide (3 μg/ mL final concentration; Sigma-Aldrich). The subculture bacterial inoculum was washed in PBSP, and 5 × 105 cfu/mL was used as a final concentration in 100 μL as final volume. The fluorescence signal (excitation 544 and emission 620 nm) was measured every 5 min for 10 h at 37 °C on a Tecan M 1000 Pro plate reader. Vancomycin Membrane Depolarization Assay. Bacterial membrane depolarization was determined by using the membrane potential-sensitive cyanine dye 3,3′-dipropylthiadicarbocyanine iodide (DiSC3(5)) (Sigma-Aldrich) as previously described with minor modifications.43 S. aureus strain ATCC 25923 was cultured as previously mentioned above, and mid log phase cells were centrifuged at 4000g for 10 min at room temperature and resuspended in assay buffer (5 mM HEPES buffer (Corning) containing 20 mM glucose pH 7.4; SigmaAldrich). The bacterial suspension was then diluted 100-fold in assay buffer (OD600: 0.005) and treated with 0.2 mM EDTA (Invitrogen, pH 8.0) to allow dye uptake. Then, 0.4 μM DiSC3(5) was added and incubated for 1 h at room temperature to allow self-quenching. DiSC3(5) bacterial uptake was monitored using Tecan M 1000 Pro plate reader (excitation 620 and emission 670 nm) with fluorescence signal measurements every 1 min. After 1 h, 0.1 M KCl (Sigma-

reduced pressure. The compound was purified by preparative HPLC to give N3-PEG3-NH-vancomycin (168 mg, 23% yield) as a white solid. Preparative HPLC was run on an Agilent Technologies (1260 Infinity) instrument using a preparative column (Agilent Eclipse XDB-Phenyl, 30 × 100 mm, 5 μm particle size) with a gradient elution (flow rate of 20 mL/min, room temperature using 0.05% formic acid in water and 0.05% formic acid in acetonitrile as eluents; gradient timetable: 5 to 50% B for 20 min, wash). Azide-vancomycin was characterized by NMR41 and LCMS (Figures S2 and S3). Conjugation and Characterization of Van-NPs. Superparamagnetic carboxylated NPs (EMD Millipore, M1-020/50) (170 nm) were coated with human serum albumin (HSA) using carbodiimide EDC (EDAC) and N-hydroxysulfosuccinimide (Sulfo-NHS) reaction according to the manufacturer. The amount of HSA was determined according to Sanjaya et al.42 Briefly, for 500 μL of carboxylated nanoparticles, 6.25 mg of both sulfo-NHS (Thermo Scientific) and carbodiimide EDC (Chem-Impex International Inc.) were dissolved in ice cold 2(N-morpholino)ethanesulfonic acid sodium salt (MES) buffer pH 5.0 (Sigma-Aldrich). After 15 min, the NPs were washed quickly in PBSP (phosphate buffered saline, 0.1% pluronic F127; Sigma-Aldrich) buffer; then, 50 mg of HSA (SigmaAldrich) dissolved in PBSP was added and incubated for 2 h at room temperature with mixing. The nanoparticles were then washed and suspended in PBSP buffer. The formed protein layer was quantified by BCA kit for quantifying proteins (Thermo Scientific). Passivated HSA-NPs were coupled with a NHS-PEG4DBCO linker (Click Chemistry tools) by reacting 500 μL of HSA-NPs (50 mg/mL) with 12.5 μL of NHS-PEG4-DBCO linker (100 mg/mL) dissolved in DMSO for 2 h at room temperature and then washed. The DBCO layer was quantified by reacting 5 μL of DBCO-NPs with 1 μL of N3-NBD fluorescent dye (Figure S5B) (0.1 mg/mL, in-house) in 1% DMSO via copper-free click chemistry through incubating them in phosphate buffer for 1 h at 37 °C. The fluorophore-coupled nanoparticles were then washed 3 times with PBSP and resuspended in 100 μL as a final volume with fluorescence measured on a Tecan M 1000 Pro plate reader at excitation of 460 and emission of 540 nm using a negative control of unconjugated nanoparticles. The washed DBCO-functionalized NPs were conjugated to different concentrations of N3-vancomycin (final concn = 0.125−3 mg/mL) in PBSP via copper-free click chemistry by incubating them together for 4 h at 37 °C. Conjugated vancomycin was quantified by fluorescent Fam-Kaa (Figure S5C) (custom synthesized by Mimotopes; 100 μg/mL in PBS) by incubating 1 μL of Van-NPs (NPs concn = 25 mg/mL) with 200 μL of Fam-Kaa (100 μg/mL) for 1 h at 37 °C with rotation. Then, the nanoparticles were washed 3 times with PBSP and resuspended in 100 μL of PBSP as a final volume with fluorescence measured on a Tecan M 1000 Pro plate reader at excitation of 500 and emission of 520 nm. Vancomycin quantification calculations were done in reference to a standard curve (Figure S6) of serial dilutions of Fam-Kaa concentration in the presence of the same concentration of NPs. Functionally active conjugated vancomycin was quantified by LCMS of eluted Ac-Kaa by incubating 25 μL of Van-NPs (NPs concn = 50 mg/mL) with 25 μL of Ac-Kaa (10 mg/mL) for 1 h at 37 °C with rotation. Then, the nanoparticles were washed 3 times with PBSP and resuspended in 50 μL of 100 G



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by BEI Resources, NIAID, NIH: NARSA STRAINS Staphylococcus aureus, Strain VRS-1, and Strain VRS-4. The authors acknowledge the facilities, and the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, The University of Queensland, with special thanks to Dr. Erica Lovas.

Aldrich) was added to equilibrate the cytoplasmic and external K+ concentrations. For membrane permeabilization to be tested, 90 μL of quenched bacterial suspension was aliquoted in 96-well black plates (Corning), and 10 μL of vancomycin, N3vancomycin, and Van-NPs (at MIC concentration) were added to each well after stabilization of fluorescence signal. The fluorescence intensity was then measured every 1 min. Triton-X (0.1%; Sigma-Aldrich), HSA-NPs, and sterile water were used as controls. In this assay, Van-NPs were prepared by using carboxylated blue FluoSpheres nanoparticles (200 nm) (Life Technology) instead of mangetic nanoparticles as previously mentioned to prevent fluorescence signal absorption due to the release of the dye. ATP Leakage Determination. Bacterial ATP leakage was determined by using an ATP determination kit (Life Technology) based on the luminescence results from firefly luciferase enzyme as previously described.36 Briefly, S. aureus strain ATCC 25923 was cultured as previously mentioned above, and mid log phase cells were centrifuged at 4000g for 10 min at room temperature and resuspended in PBS buffer. Bacterial cells (∼108 cfu/mL) were incubated with vancomycin and different densities of Van-NPs at MIC concentration (final volume = 100 μL) for 1.5 h. Cells were removed by centrifugation at 4000g for 10 min at room temperature, and supernatants were assessed for ATP concentration using Tecan M 1000 Pro plate reader with an integration time of 1000 ms. Quantification was done in reference to a standard curve (Figure S10).

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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.6b00494. Experimental details and characterization of N3-vancomycin, N3-NBD, HSA-NPs, Fam-Kaa, Ac-Kaa, and VanNPs (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Marwa M. Hassan: 0000-0001-9629-3802 Mark A. T. Blaskovich: 0000-0001-9447-2292 Matthew A. Cooper: 0000-0003-3147-3460 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to acknowledge the University of Queensland UQ ECR 2015 grant and NHMRC Project Grants APP631632 and APP1026922 for partially funding this work. M.A.C. is a National Health and Medical Research Council (NHMRC) Principal Research Fellow (APP1059354) and former Australia Fellow (AF511105). M.A.T.B was supported in part by a Wellcome Trust Seeding Drug Discovery Award 094977/Z/10/Z, W.P. by an Australian Postgraduate Award (APA) Ph.D. scholarship, and M.M.H. by the University of Queensland International Ph.D. scholarship. The following reagent was provided by the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for distribution H



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Surface Ligand Density of Antibiotic-Nanoparticle Conjugates

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