From Structure to Function: pH-Switchable Antimicrobial Nano-Self

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Biological and Medical Applications of Materials and Interfaces

From Structure to Function: pH-Switchable Antimicrobial Nano-Self-Assemblies Mark Gontsarik, Anan Yaghmur, Qun Ren, Katharina Maniura-Weber, and Stefan Salentinig ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18618 • Publication Date (Web): 27 Dec 2018 Downloaded from http://pubs.acs.org on January 1, 2019

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ACS Applied Materials & Interfaces

From Structure to Function: pH-Switchable Antimicrobial Nano-Self-Assemblies Mark Gontsarika, Anan Yaghmurb, Qun Rena, Katharina Maniura-Webera, Stefan Salentiniga* aLaboratory

for Biointerfaces, Empa Swiss Federal Laboratories for Materials Science and Technology, Lerchenfeldstrasse 5, 9014, St. Gallen, Switzerland

bDepartment

of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark.

KEYWORDS: antimicrobial nanomaterials, self-assembly, antimicrobial peptide delivery, amphiphilic lipids, pH-triggered nanocarriers, E. coli., micelles.

ACS Paragon Plus Environment

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ABSTRACT

Stimuli-responsive nanocarriers based on lipid self-assemblies have the potential to provide targeted delivery of antimicrobial peptides, limiting their side effects while protecting them from degradation in the biological environments. In the present study, we design and characterize a simple pH-responsive antimicrobial nanomaterial, formed through the self-assembly of oleic acid (OA) with the human cathelicidin LL-37 as model for an amphiphilic antimicrobial peptide. Colloidal transformations from core-shell cylindrical micelles with a cross-section diameter of ~ 5.5 nm and a length of ~ 23 nm at pH 7.0, to aggregates of branched thread-like micelles at pH 5.0 were detected using synchrotron small angle X-ray scattering, cryogenic transmission electron microscopy, and dynamic light scattering. Biological in vitro assays using an Escherichia coli bacteria strain showed high antimicrobial activity of the positively charged LL37/OA aggregates at pH 5.0, which was not caused by the pH conditions themselves. Contrary to that, negligible antimicrobial activity was observed at pH 7.0 for the negatively charged cylindrical micelles. The nanocarrier’s ability to switch its biological activity ‘on’ and ‘off’ in response to changes in pH could be used to focus the antimicrobial peptides’ action to areas of specific pH in the body. The presented findings contribute to the fundamental understanding of lipid-peptide self-assembly, and may open up a promising strategy for designing simple pHresponsive delivery systems for antimicrobial peptides.


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INTRODUCTION

Antimicrobial peptides (AMPs), including the only human cathelicidin LL-37, are emerging as promising alternatives to conventional antibiotics.1-2 However, they suffer from chemical instability in biological environments, and may cause host toxicity at higher concentrations, leading to a significant reduction in their therapeutic efficiency.3 In light of the global surge of antibiotic resistance among human-pathogenic bacteria, the recent and increasing interest in the development of nanoparticles for pharmaceutical delivery of AMPs is thus well founded.4 Self-assembled structures, including micelles, microemulsions and inverse lyotropic nonlamellar liquid crystalline phases, have attracted increasing attention for their potential applications in drug delivery.4-11 Formed by the self-assembly of biologically relevant amphiphilic lipids in excess water, these structures are in thermodynamic equilibrium with their surroundings and provide large lipid-water interfacial area for the solubilization of various bioactive molecules such as proteins and peptides or host them in their aqueous or oil domains.1218

Encapsulation of guest molecules into the nanoparticle dispersions of such nanostructures may

offer many advantages including protection from proteolysis and oxidation.11, 19-23 Additionally, such self-assembled nanoobjects can be designed to be responsive to various stimuli including temperature,24-25 light,26-27 presence of enzymes,28-29 and pH.30-33 These stimuli trigger structural changes in these colloidal nanoparticles and selectively modulate their activity, or facilitate release of their active cargo. In this context, pH-sensitive lipid-based nanocarriers for AMPs are attractive for a wide range of pharmaceutical applications as they could provide self-regulated passive targeting to areas of specific pH in the body.34 However, their further design requires improved understanding of the physico-chemical interactions involved in the AMP/lipid self-


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assembly, and the effect of pH on modulating their structural features, along with their biological activity. Previously, our research group has reported on the integration of LL-37 into lipid nanocarriers based on glycerol monooleate (GMO)15 and oleic acid (OA).32 Spontaneous transfer of LL-37 into the lipid nanostructures was observed as the amphiphilic nature of the peptide’s α-helical structure allowed its integration and active participation in the self-assembly of both lipids, modulating their nanostructures in a concentration-dependent manner.15,

32

Strongly dependent

on the type of structure, the LL-37/GMO nano-self-assemblies were found to have variable antimicrobial activity.15 Encapsulated in the internal bicontinuous cubic nanostructure of GMO cubosomes, the AMP did not exert significant bactericidal effects on Escherichia coli (E. coli), whereas in form of vesicles and micelles, the AMP-loaded nanocarriers had a stronger and more rapid antimicrobial effect than the AMP by itself, free in solution.15 Hence, designing LL37/lipid nanocarriers with pH-sensitive structures could potentially allow on-demand switching between biologically active and inactive nanostructures through pH-triggered phase transformations. Recently, the protonation state of the OA’s carboxylic group at different pH was found to have a strong effect on the molecular lipid-peptide packing in the produced LL-37/OA nanostructures.32 At pH below 7.0, the formation of nanoparticles enveloping an inverse micellar cubic Fd3m phase, emulsified microemulsions with an internal L2 phase, and oil-inwater (O/W) emulsions was favored, while at pH above 7.5 hydrophilic structures including normal cylindrical micelles and vesicles were formed.32 These pH-sensitive AMP/OA nanostructures were also observed to be composition-dependent as the integration of LL-37 at the lipid-water interface caused significant electrostatic repulsions among the cationic AMP molecules, favoring the formation of more hydrophilic structures.32


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The structural sensitivity of LL-37/OA nanocarriers to pH could be relevant for treating bacterial infections as the glycolytic metabolism of certain bacteria may acidify the infected locations.35 These local pH environments could trigger the necessary structural changes to activate the bactericidal effects of the AMP/lipid nanocarriers locally, potentially leading to an improved therapeutic efficiency owing to a more site-targeted peptide activity. In addition, such pH-switchable antimicrobial systems may be beneficial for the treatment of bacterial infections in the gastro-intestinal tract, for instance, in the treatment of gastric ulcers by focusing the antimicrobial activity to the low pH environment of the stomach, while sparing the probiotic bacteria in the elevated pH conditions of the intestine.36-38 The current study presents new design and in-depth characterization of pH-responsive LL37/OA nanocarriers based on the self-assembly of OA with LL-37. Synchrotron small angle Xray scattering (SAXS), dynamic light scattering (DLS) and cryogenic transmission electron microscopy (cryo-TEM) are used to investigate the effect of pH on the nanostructure of these self-assemblies and in vitro biological assays are conducted to correlate the nanostructure with the bactericidal activity against E. coli. The results show pH-switchable biological activity of AMP-loaded nanocarriers and could pave the way for the improvement of AMP’s therapeutic efficiency through focusing their activity to local physiological pH environments.

EXPERIMENTAL SECTION Nanocarrier preparation. Oleic acid (OA, purity ≥ 99%, Sigma-Aldrich, Buchs, Switzerland) was mixed with 0.02 M phosphate buffer saline (PBS, see SI for details on preparation) at pH 8.0 at a final OA concentration of 20 mg mL-1 and homogenized by ultrasonication with a tip sonicator (Sonics Vibra Cell VCX 130 W, 20 kHz, Sonics & Materials Inc., Newton, CT, USA)


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for 1 min (1 s pulse, 1 s break) at max. amplitude of 20%. Thereafter, the pH of the OA dispersion was readjusted to 8.0 with 1 M NaOH (≥99% purity, Carl Roth GmbH, Karlsruhe, Germany). These samples were then further sonicated for 2 min (3s pulse, 5s break) at 27% of maximum amplitude, resulting in a homogeneous, opaque dispersion. The LL-37/OA nanocarriers were then prepared by first dissolving 20 mg mL-1 LL-37 (trifluoroacetate salt, 99,18% purity, CASLO ApS, Lyngby, Denmark) in PBS at pH 8.0. This LL-37 solution was then mixed with the OA dispersion at a LL-37/OA mass ratio of 8/2. The resulting concentration of LL-37 and OA in the mixture was 16 mg mL-1 and 4 mg mL-1, respectively. The pH of the nanocarrier solution was adjusted to the selected values using 1 M HCl and 1 M NaOH and the samples were equilibrated at room temperature for at least 30 min prior to further experiments. The 1 M HCl solution was prepared from 37% HCl stock (ACS reagent grade, Sigma-Alrdich, Buchs, Switzerland) by dilution with water. Ultra-pure water (resistivity >18 MΩ cm) was used for the preparation of all samples. Samples were further diluted for microbiological assays and ζ-potential measurements, and pH of the samples were measured and, if necessary, readjusted to defined values after dilution. Cryogenic transmission electron microscopy (cryo-TEM). A 3 µl drop of the sample was placed on a Lacey Formvar holey film with a lacey structure enforced by a silicon monoxide coating, supported by a TEM copper grid (300 mesh, Ted Pella Inc., Redding, CA, USA) that was previously subjected to glow discharge treatment (Leica Inc. EM ACE 200, Germany) to ensure better settling of the sample onto the grid. Excess liquid on the grid was removed by blotting with absorbing filter paper for 5 s and the sample was rapidly plunged into liquid ethane cooled close to its freezing point by liquid nitrogen (-180 °C) (FEI Vitrobot IV, Holland). The vitrified samples were transferred with a Gatan 626 cryoholder (Gatan Inc., Pleasanton, CA,


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USA) and analyzed with Tecnai G2 20 transmission electron microscope (FEI, Holland) equipped with a FEI Eagle 4 x 4k CCD camera at 200 kV under low-dose conditions (~ 10 e Å2s-1)

at x29000 magnification. Crystalline artifacts could potentially be present in the vitrified

films.39 Images of the sample at pH 4.0 are not shown due to unsuccessful attempts of its vitrification prior to cryo-TEM observations. For the size estimation of the population of the small aggregates of branched micelles formed at pH 5.0, the Fiji extension of the ImageJ program was used.40 The images were initially smoothed, the background was subtracted (50 pixel ball radius), and a threshold was applied to generate a binary image of the nanoparticles. The particle size was estimated by fitting ellipsoids to aggregates from 2 different images for the same sample and averaging the lengths of the major and minor axes of the ellipsoids from both images. Presence of the small aggregates was observed in majority of the images taken. Dynamic Light Scattering (DLS) and ζ-potential measurements. The hydrodynamic radius (RH), polydispersity index (PDI), and ζ-potential were measured using Malvern Zetasizer Nano ZS90 (Malvern Instruments, USA) with a He-Ne Laser beam at a wavelength of 633 nm, laser power of 4 mW at 25° C. The LL-37/OA dispersions were used for size characterization at a scattering angle of 90°, after centrifugation at 3000 g for 10 min to remove dust and large aggregates. RH was estimated from the apparent diffusion coefficient, Dapp, obtained by the cumulant analysis of the correlation function,41 by using the Stokes-Einstein equation: 𝑅𝐻 =

𝑘𝐵𝑇

(1)

6𝜋𝜂𝐷𝑎𝑝𝑝

where kB is the Boltzmann constant, T is the absolute temperature, and η is the viscosity of the solvent (water). PDI was estimated from the second cumulant of the correlation function as following:


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𝑃𝐷𝐼 =

𝜇2

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(2)

𝛤

where μ2 is the second cumulant, and 𝛤 is the mean of the inverse decay time. In addition, the volume-weighted particle size distribution was calculated from the correlation functions by the Zetasizer Software 7.11 (Malvern Instruments, USA). It should be noted that the presented RH are apparent values due to the elongated shape of the elongated shape of the nanocarriers and the measurements being performed without further dilution of the sample. For ζ-potential measurements the samples were diluted 1/10 with PBS and their electrophoretic mobility, μe, was measured in DTS1070 capillary cells (Malvern Instruments Ltd., UK). ζpotential is calculated using Smoluchowski theory: 𝜇𝑒 =

𝜀𝑟𝜀0𝜁

(3)

𝜂

where εr is the dielectric constant of water, ε0 is the permittivity of vacuum, and η is the viscosity of water. All measurements were done in triplicates and presented as an average. Synchrotron small angle X-ray scattering (SAXS). Structural characterization experiments were performed in part at the I22 SAXS beamline (Diamond Light Source synchrotron facility, Oxfordshire, UK), and in part at the cSAXS beamline (Swiss Light Source at Paul Scherrer Institute, Villigen, Switzerland) due to the loss of beam during the measurements. Measurements at the I22 SAXS beamline were conducted at an operating electron energy of 12.4 keV and X-ray wavelength of 0.9999 Å. Sample to detector distance of 3563 mm provided a q range (q = 4π/λ sin θ, where λ is the X-ray wavelength, and 2θ is the scattering angle) of 0.08–6.1 nm-1 and a beam size of 120 x 370 μm. The exposure time was 5 s and the measurements were done in triplicate and presented as an average.


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Measurements at the cSAXS beamline were done at an operating electron energy of 11.2 keV with X-ray wavelength of 1.1079 Å, a sample to detector distance of 2152 mm, and q-range of 0.08–5.8 nm-1, and a beam size of 290 x 360 μm. The exposure time was 1 s and the measurements were done in triplicate and presented as an average. After pH adjustment, the samples were loaded into borosilicate glass capillaries, and 2-D scattering patterns were collected on a Pilatus3 2M detector with an active area of 254 x 289 mm2 and 172 x 172 μm2 pixel size (Dectris, Baden, Switzerland) within an hour after preparation at both beamlines. The sample holder was equipped with a water jacket, and the measurements were done at 37 °C. The 2D scattering frames were radially integrated into 1D curves and plotted as a function of relative intensity, I(q) versus q. Scattering from PBS was subtracted from all samples as background. SAXS data analysis. The cylindrical micelles were identified by their q-1 dependence of I(q) at low q values, characteristic of scattering from rod-like objects. Additionally, the generalized indirect Fourier transformation method (GIFT) was used to analyze the SAXS data and estimate the pair-distance distribution functions p(r). This method can separate the scattering intensity contributions of N number of monodisperse, homogenous, and spherical particles into two factors42-43: 𝐼(𝑞) = 𝑁𝑆(𝑞)𝑃(𝑞)

(4)

where the P(q) describes the form factor, and S(q) the structure factor. A model for the structure factor is selected and fitted simultaneously with the form factor to the experimental data using equation 4. Considering the charged nature of the nanoparticles, the chosen structure factor


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model was based on Ornstein-Zernike equation with the screened Coulomb interaction potential and using hypernetted-chain approximation as a closure relation.44 The 𝑝(𝑟) is calculated from the form factor P(q) using equation 5:42-43

∫

∞

𝑃(𝑞) = 4𝜋

𝑝(𝑟) 0

sin 𝑞𝑟 𝑑𝑟 𝑞𝑟

(5)

For particles of arbitrary shape, the 𝑝(𝑟) is given by 𝑝(𝑟) = 𝑟2∆𝜌2(𝑟), where ∆𝜌2(𝑟) is the convolution square of the excess electron density distribution, ∆𝜌(𝑟), averaged over all directions in the space. For cylindrical particles of length L, the scattering intensity I(q) can be expressed as: 𝐼(𝑞) =

𝐿𝜋 ∙ 𝐼𝑐(𝑞) 𝑞

(6)

The cross-section scattering function Ic(q) is related to the cross-section distance distribution function 𝑝𝑐(𝑟) by Hankel transformation:

∫

∞

𝐼𝑐(𝑞) = 2𝜋

𝑝𝑐(𝑟)𝐽0(𝑞𝑟)𝑑𝑟

(7)

0

where J0(qr) is the Bessel function of the zero order. For cylindrical symmetry, 𝑝𝑐(𝑟) is 2

2

described by 𝑝𝑐(𝑟) = 𝑟 ∙ 𝜌𝑐 (𝑟), where 𝜌𝑐 (𝑟) is the convolution square of the radial excess electron density distribution function, 𝜌𝑐(𝑟𝑐), with 𝑟𝑐 being the normal distance to the cylinder axis. The radial profile 𝜌𝑐(𝑟𝑐) was obtained from 𝑝𝑐(𝑟) by the convolution square root operation performed with DECON program.45-46 The inter-lamellar distance d for multilamellar vesicles was calculated using 𝑑=

2𝜋ℎ 𝑞ℎ

(8)

where h is the order of the Bragg peak, and qh is the q-value of the h-th order Bragg peak.


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In vitro antimicrobial microdilution plating assay. The bactericidal activity of LL-37/OA nanocarriers at pH 5.0 and 7.0 was investigated using the E. coli strain U5/41T (DSM 30083 urine isolate, DSMZ, Braunschweig, Germany) using the microdilution plating assay47-48 similar to a previous study.15 Glycerol stocks of E.coli were spread-plated on Plate Count (PC) Agar (Sigma-Aldrich, Buchs, Switzerland) and incubated overnight. A few of the emerging single colonies were transferred after to a 25 vol% dilution of brain-heart-infusion (BHI) medium (Sigma-Aldrich, Buchs, Switzerland) in PBS at pH 5.0 or 7.0 for an overnight incubation. To test the antimicrobial activity of nanocarriers against bacteria in their mid-exponential growth phase and to minimize the fraction of dead cells present in the culture through dilution, on the day of the experiment 0.5 mL of each of the overnight cultures was used to inoculate 4.5 mL of fresh 25 vol% BHI medium of the same pH and was further incubated. Before the antimicrobial experiments, the optical density OD600 of these freshly inoculated 25 vol% BHI media was measured over 24 hours using a Synergy H1 UV spectrophotometer (BioTek Instruments GmbH, Luzern, Switzerland) at both pH 5.0 and 7.0 to monitor bacterial growth. The E.coli in this fresh inoculate were observed to be in their exponential growth phase for at least the first 3.5 h. Therefore, the last incubation step was chosen to last no longer than 3 h. All incubations took place at 37 °C on an orbital shaker at 60 rpm. After incubating the fresh 25 vol% BHI inoculates for up to 3 hours, 1 mL of these cultures, still in their exponential growth phase, were transferred to 2 mL Protein LoBind Tubes (Eppendorf AG, Hamburg, Germany) and centrifuged at 13000 x g for 3 min (miniSpin plus, Vaudaux-Eppendorf, Schönenbuch, Switzerland) to pellet the bacteria. The supernatant was removed and the pellet was re-suspended with fresh 1 vol% BHI solution in PBS at the same pH. 1 vol% BHI solution was chosen as growth media because it sustained the bacterial growth rate


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required for the experiment. The optical densities (OD595) of the bacterial dispersions in 1 vol% BHI solution were then measured (BioPhotometer plus, Eppendorf AG, Hamburg, Germany) to estimate their E.coli concentrations according to a calibration curve. The calibration curve was measured beforehand by diluting the bacterial dispersions with 1 vol% BHI solution, measuring each of their OD595 and then quantifying the E.coli populations by the microdilution plating method described below. According to the OD595 values, the 1 vol% BHI dispersions were then diluted with 1 vol% BHI to have 5.5 × 105 colony forming units (CFU) per mL and used as a starting population in the antimicrobial experiments. 900 μL of the 1 vol% BHI culture containing 5.5 × 105 CFU mL-1 at pH 5.0 and 7.0 were transferred into 2 mL Protein LoBind Tubes (Eppendorf, Hamburg, Germany) and treated with 100 μL of LL-37/OA solutions adjusted to the same pH. LL-37 and OA final concentrations in the culture medium were 80 and 20 μg mL-1, respectively. The experiments were repeated with PBS only and 20 μg mL-1 OA as negative controls, and 80 μg mL-1 LL-37 as a positive control, at both pH 5.0 and 7.0. For these experiments, the samples were kept at 37 °C under gentle shaking at 500 rpm (Thermomixer comfort, Vaudaux-Eppendorf AG, Basel, Switzerland). To quantify the viable bacterial populations over the duration of the experiment using the microdilution plating assay,15, 47-48 50 μL fractions of the bacterial suspensions were taken out after 15, 30, 60, 120, 180 min of treatment and serially diluted 10-fold with PBS of the same pH 5 times. 5 μL from each of the resulting dilutions were plated in triplicates on PC Agar. The total time from the removal of the sample from the reaction vessel to plating it onto the agar plate was kept below 5 min. After overnight incubation at 37 °C, the CFU were quantified from the distinguishable colonies formed on the plates with Scan 300 automatic colony counter (Interscience, Saint Nom, France). Each experiment was repeated thrice, and the obtained results


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were presented as an average and standard deviation of the log differences in the bacterial population Nt at time t from the starting population N0 of 5.5 × 105 CFU mL-1 (calculated as log(Nt /N0)). GraphPad Prism software (GraphPad Software, USA) was used for student’s t test statistical analysis (p < 0.05). After the last sample extraction, the remaining bacterial culture was filtered using 0.2 μm pore size filters (Titan2 Syringe Filter, PES, Thermo Scientific Sun SRi, USA), and had its pH measured to ensure that the conditions stayed constant throughout the experiment. Only negligible differences in pH of about ± 0.3 pH units were observed.

RESULTS AND DISCUSSION Figure 1 shows the SAXS curves from pH-dependent nanostructures of LL-37/OA selfassemblies at LL-37/OA weight ratio of 8/2. At pH 6.0–8.0, the SAXS data show a q-1 dependence in the q-range of 0.1-0.4 nm-1 (Figure 1a), indicating presence of cylindrical micelles, and a diffuse correlation peak between q of 1.3 and 3 nm-1 that is most likely attributed to scattering from the cylinder cross-section. The generalized indirect Fourier transformation method (GIFT) was employed to analyze the experimental SAXS profiles and gain further insight into the structural features of LL-37/OA nanoobjects. From GIFT analysis (Figure 1b,c), the shape of the pair-distance distribution function, p(r), calculated for the sample at pH 7.0 is characteristic of core-shell type cylinders with a local minimum in the p(r) at r ≈ 2 nm (Figure 1c), arising from a difference in excess electron density between the core and the shell. In addition, a gradual decrease in the p(r) at r > 6 nm was observed indicating an elongated structure with a maximum dimension of ~ 23 nm, estimated from p(r) = 0 (see Figure 1). Considering the cylindrical symmetry of the self-assemblies, the cylindrical cross-section pairdistance distribution function, 𝑝𝑐(𝑟), was also calculated, indicating a cylinder diameter of 5.5


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nm estimated from Dmax at 𝑝𝑐(𝑟) = 0 (see Figure 2a,b). The convolution square root operation on the 𝑝𝑐(𝑟) function was used to estimate the radial excess electron density distribution, 𝜌𝑐(𝑟𝑐), of the cylinders, further supporting the formation of core-shell structure with a core-radius of ~ 1.1 nm and a shell-thickness of ~ 1.4 nm (Figure 2c). Considering that the hydrocarbon tails of OA could make up the less electron-dense core of the cylindrical micelle and the amphiphilic LL-37 molecules are most likely to be positioned at the OA lipid-water interface,32 the ~ 1.4 nm thick shell of the high excess electron density, observed from the 𝜌𝑐(𝑟𝑐) in this study, is likely to consist of LL-37 α-helices, with an estimated average outer diameter of about 1 nm, combined with the hydrophilic headgroups of OA molecules.49-50


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Figure 1. a) SAXS curves of the LL-37/OA self-assemblies in excess PBS at pH values between 4.0 and 8.0 and LL-37/OA weight ratio of 8/2. The black dashed line and black solid


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lines were calculated to show the q-2.4 and q-1 dependence of the I(q) in those regions, respectively. The indication of a Bragg peak from potentially weakly correlated lamellar stacks at pH 4.0 is marked with an asterisk (*). The proposed structures of LL-37/OA cylindrical micelles at 7.0 and aggregates of branched cylindrical micelles at pH 5.0 are shown as graphical representations with LL-37 in green, hydrophilic headgroups of OA in blue and hydrophobic tails of OA in yellow. b) Scattering curve (symbols) and fit calculated with the GIFT method (red curve) for the sample at pH 7.0. For the structure factor parameters see Table S1. c) The corresponding p(r) calculated from the I(q) presented in b) by using equations 4 and 5.


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Figure 2. a) Experimental SAXS curve (symbols) and calculated model-dependent fit for the cylindrical cross section (red curve) for the LL-37/OA sample at pH 7.0. For structure factor parameters see Table S1. b) The corresponding cross-section pair-distance distribution function, 𝑝𝑐(𝑟), of the cylinder (red curve) calculated from the fit in a) by using equations 6 and 7, and fit


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calculated by deconvolution using a convolution square root operation (dashed black line). c) The corresponding radial excess electron density profile of the cylindrical cross-section, 𝜌𝑐(𝑟𝑐), calculated from the fit in b). The inset shows the graphical representation of the cylinder and the cross-section radius rc.

The cryo-TEM images of the LL-37/OA dispersions confirm the SAXS results: the images taken at pH 7.0 (Figure 3b), and pH 6.0 (Figure S1) show thin wormlike objects. The observed diameter of the elongated and branched structures of ~ 5 nm in the cryo-TEM images correlates well with the Dmax of ~ 5.5 nm from the pc(r) at pH 7.0 for this sample. The observed selfassembly of LL-37/OA dispersions into cylindrical micelles at pH 6.0-8.0 is most likely due to the presence of high amounts of LL-37 at the OA-water interface, which would increase the electrostatic repulsions among the cationic peptide molecules.32 In terms of the critical packing parameter (CPP, see SI for definition), this would result in an increase of the effective headgroup area at the OA-water interface leading to a decrease in the CPP and favor, therefore, the formation of more hydrophilic structures such as normal cylindrical micelles that were observed at this pH range. Compared to the results in the literature that report on the formation of cylindrical micelles at pH > 10 in OA samples without LL-37,51-52 or at pH 8.5 in LL-37/OA dispersions with lower LL-37 content of 30 wt%,32 the present study shows that these cylindrical micelles occur at lower pH (pH in the range of 6.0–8.0) upon increasing LL-37 content to 80 wt% in the binary LL-37/OA mixture. The measured ζ-potential of the LL-37/OA nanocarriers increased from -10.1 to +4.6 mV upon a decrease in the pH from 8.0 to 6.0 (Figure 4) due to the protonation of the carboxylic groups of OA at lower pH. The apparent pKa (pKaapp) of OA in the LL-37/OA self-assemblies containing up to 20 wt% LL-37 was recently reported to be around


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7.8.32 It was proposed that the deprotonation of most carboxylic groups of OA that occur around that pH value leads to major colloidal transformations.32 However, it was also reported that the interactions among LL-37 and OA molecules in excess buffer at a higher LL-37 content favor a more gradual deprotonation behavior, resulting in ζ-potential changes over a larger pH range.32 Although the major cause of pH-induced colloidal transformations in LL-37/OA self-assemblies is most likely attributed to a change in the protonation state of OA,32 the retained cylindrical micellar structure at pH 6.0-8.0, as shown in the present work, indicates that the differences in the lipid-water curvature of the LL-37/OA nanostructures caused by the protonation of OA may not be sufficient to induce a phase change within that pH range. The gradual protonation of the lysine and arginine side chains in LL-37 could further contribute to the observed increase in the ζ-potential at decreasing pH.53


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Figure 3. Representative cryo-TEM images of LL-37/OA nano-self-assemblies dispersed in PBS. a) At pH 5.0, mostly polydisperse particles with diameter of 14.9 ± 6.1 nm (marked with asterisks), potentially aggregates of branched wormlike micelles, and b) at pH 7.0, thin, branched cylindrical micelles with cross-section diameter of about 5 nm were observed.


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Figure 4. ζ-potential measurements of the LL-37/OA dispersions in PBS at pH 4.0–8.0.

For the sample at pH 5.0, the scattering intensity at q < 0.2 nm-1 shows a steep upturn, potentially caused by the scattering from particles with sizes beyond the SAXS experimental resolution limit (Figure 1a). The diffuse correlation peak in the q range of 1–3 nm-1 in that curve could be from the cross-sectional scattering from the cylindrical micelles in the larger aggregates. This correlation peak at pH 5.0 appears less pronounced and slightly shifted to lower q values compared to pH 8.0-6.0 which may result from the increased number of junctions on the branched cylindrical micelles. The cryo-TEM images of the same sample are dominated by a population of defined nano-objects with an estimated diameter of 14.9 ± 6.1 nm (Figure 3a), potentially condensed branched thread-like micelles coexisting with some larger aggregates up to the micrometer range (Figure S2). A representative close-up image of the larger aggregates in the sample (Figure S2) showed striated patterns in the interior of the particle, in agreement with the observation of aggregates of thread-like micellar structures as indicated by SAXS. The thin elongated protrusions within this particle are roughly 5 nm in thickness, similar to the diameter of LL-37/OA cylindrical micelles formed at pH 6.0–8.0. The colloidal transformation of the branched LL-37/OA cylindrical micelles at pH 6.0 to aggregates of variable size at pH 5.0 is thought to be triggered by the modified protonation of OA. The decrease in the fraction of deprotonated anionic OA molecules with decreasing pH


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reduces the electrostatic repulsions among the OA headgroups, which would result in a decrease of the effective headgroup area at the OA-water interface and lead to an increase in the CPP. The associated decrease in the positive lipid-water interfacial curvature of the cylindrical micelles makes the formation of junctions energetically more favorable and can result in their branching.54 At pH 4.0, the shoulder at q in the range of 1.3–3 nm-1 diminished in intensity, and the scattering intensity showed a q-2.4 dependence at q ≤ 0.9 nm-1, suggesting a transition to lamellar structures such as vesicles.55 At this pH, the appearance of a Bragg peak at q around 1.0 nm-1 could represent the formation of weakly ordered lamellar structures. Taking into account the thickness of the water layer between these highly disordered lamellar structures, the interlamellar distance of around 6.3 nm, which was calculated from this very weak peak by applying equation 8, presumably correlates with the previously reported bilayer thickness of 4.9 nm for LL-37-free pristine OA vesicles.56 This transition may result from the further OA protonation and increase of the CPP in the system, potentially reaching unity and thus favoring formation of lamellar structures. Such transitions from cylindrical micelles to lamellar structures upon flattening of the lipid-water interfacial curvature have been previously reported in surfactant selfassemblies.57-59 The DLS measurements of the LL-37/OA dispersions at pH 8.0, 7.0 and 6.0, showed a correlation function with a single relaxation time only (Figure S3a) with corresponding apparent hydrodynamic radii of 5.1 nm (PDI = 0.086), 5.1 nm (PDI = 0.061), and 5.3 nm (PDI = 0.039) from cumulant analysis, respectively. The DLS correlation functions for the samples at pH 5.0 and 4.0 indicate multimodal particle size distributions, suggesting the formation of branched thread micelles coexisting with larger aggregates with internal thread micelles. The volume-


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weighted particle size distribution functions calculated for these polydisperse samples indicate that a larger fraction of the particles is still around 5 nm (Figure S3b). However, the centrifugation step prior to DLS measurement might have led to a higher weighting of the small particles by removing some of the larger aggregates from the scattering volume. Overall, the formation of larger particles upon lowering pH, observed by DLS, correlates well with findings from SAXS and cryo-TEM that indicate the formation of aggregates of branched cylindrical micelles at pH 5.0. The cryo-TEM image in Figure S1, taken for the same sample after increasing pH from 5.0 to 6.0, suggests that the structural features are reversible on circulating pH. The pH triggered ‘on’ / ‘off’ switch of the antibacterial activity of the LL-37/OA nanocarriers was investigated using microdilution plating method.47 Figure 5 compares the viable populations of E. coli bacteria at pH 5.0 and pH 7.0 after treatment with free LL-37, free OA, and LL-37loaded OA nanocarriers over a period of 180 min. After treatment with the LL-37/OA nanocarriers at pH 5.0 (‘on’-switch), the viable E. coli population sharply declined, reaching complete elimination after 180 min. However, the treatment with the same LL-37/OA nanocarrier at pH 7.0 (‘off’-switch) left the bacterial growth mostly unaffected: compared to the control sample with PBS only, the differences in bacteria numbers did not exceed 0.5 log10 over the 180 min duration of the treatment. As the pH of the culture media may also influence the growth of the bacteria, treatment with only PBS was studied as negative control at pH 5.0 and 7.0. The bacteria in the negative control group were able to sustain the same exponential growth rate at both pH values within the time scale of the antimicrobial experiments, considering the inherent inaccuracy of ± 0.5 log10 in population estimation for such an experiment.48 Also free


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OA had no significant effect on the growth of E. coli within this pH range (student’s t test p > 0.05).

Figure 5. pH-dependent bactericidal activity of the LL-37/OA nanocarriers containing 80 μg mL-1 LL-37 and 20 μg mL-1 OA against E. coli at pH 5 and pH 7 (red curves). The nanocarriers were antimicrobially active at pH 5.0 (red full triangles and dashed line), but had no bactericidal activity at pH 7.0 (red full circles and full line). PBS and 20 μg mL-1 OA were used as negative controls (black and green symbols) and did not show antimicrobial activity pH 5.0 and 7.0 (student’s t test p > 0.05). Free LL-37 at 80 μg mL-1 used as positive control was antimicrobially active at both pH 5.0 and 7.0 (blue symbols).

Upon treating the bacterial cultures with free LL-37 as positive control, the bacteria were eradicated at both pH 5.0 and 7.0, reaching complete elimination of E.coli after 60 min at pH 7.0 and 180 min at pH 5.0. This is in line with the previously reported antimicrobial activity of LL37 against this E. coli strain at pH 6.5.15 Interestingly, the LL-37/OA nanocarriers at pH 5.0 eliminated significantly more bacteria than free LL-37 at the same pH in the first 30 minutes of


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the treatment (student’s t test p < 0.05, the difference exceeded 0.5 log10) indicating a somewhat faster killing of the bacteria. This could result from the improved mass transfer of LL-37 to the bacteria membrane by shuttling of multiple LL-37 embedded in the nanocarriers. Previous reports also suggested that the local LL-37 concentration at the bacterial membrane needs to exceed a certain thresholds in order to exert destructive effect.60-61 After 60, 120 and 180 min of treatment no significant differences (student’s t test p < 0.05) were observed between the E.coli populations in the groups treated with LL-37/OA and free LL-37 at pH 5.0, potentially indicating that the membrane was saturated with LL-37 at those time points. The presence of proteins in the BHI media appear not to interfere with the antimicrobial activity of the presented nanocarriers. The switch in the antimicrobial activity of the LL-37/OA nanocarriers may be related to pH induced modifications in their surface charge and nanostructure. The nanocarriers had positive ζpotential at pH 5.0, which could trigger electrostatic attractions between the nanocarriers and the negatively charged bacterial cell membrane. Contrary, the cylindrical micelles at pH 7.0 showed negative ζ-potential, potentially causing repulsion between the anionic bacterial membrane and the nanocarriers. The pH-dependent modification of the curvature of the lipid-water interface may also contribute to the nanocarrier – bacteria interactions and related antimicrobial activity. The interfacial curvature of the flat bilayer membrane of the bacteria is significantly different to the curved cylindrical LL-37/OA micelles at pH 7.0, whereas this difference is decreasing for branched cylindrical micelles and even more for lamellar structures such as vesicles. The demonstrated ‘on’ / ‘off’ switch of the LL-37/OA nanocarriers’ antimicrobial activity between pH 5.0 and 7.0 could potentially be of interest for targeted peptide delivery to areas in the body with specific pH conditions, while minimizing activity in other sites. For example, the treatment of gastric ulcers with conventional antibiotics against the helicobacter pylori bacterial


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infection is commonly associated with elimination of the commensal bacterial populations in the intestine, which are essential for the maintenance of human health.38,

62-63

The nanocarriers

presented here could potentially exert bactericidal activity under the acidic conditions of the stomach, leading to the successful treatment of the mentioned infection, while later switching to their antimicrobially inactive form in the slightly basic conditions of the intestine,36-37 which could lead to sparing of the probiotic populations of the commensal bacteria. pH-sensitive changes in the antimicrobial activity of LL-37/OA nanocarriers could potentially minimize the exposure of LL-37 to healthy cells and limit, therefore, its side effects.64

CONCLUSIONS A simple and versatile nanocarrier system based on the self-assembly of LL-37 with OA was designed with the ability to switch between its antimicrobially active and inactive states through pH-triggered structural transformations. Upon decreasing the pH from 8.0 to 4.0 in this system, transformations from cylindrical micelles to branched thread micelles coexisting with larger particles with internal thread micelles and lamellar structures were observed. These nanostructural transformations are triggered by the gradual protonation of OA with decreasing pH and agree well with the considerations from the critical packing parameter model. The protonation of OA upon decreasing pH in the LL-37/OA nanocarriers leads to a loss in the negative charge at its carboxylic headgroup, and thus results in a decrease in the effective headgroup area at the lipid-water interface, increasing the CPP from < 1 to around unity. Antimicrobial studies using the worm-like micelles at pH 7.0 indicated negligible activity against E. coli, potentially attributed to their structure combined with the electrostatic repulsions between the negatively charged micelles and the anionic bacterial membrane. Contrary, the


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branched thread micelles and aggregates at pH 5.0 were highly active against the bacteria in our in vitro model. The modified nanostructure and their positive surface charge are thought to facilitate the interactions with the negatively charged bacterial membranes. The novel pH-responsive antimicrobial nanocarriers designed in this study are a promising candidate for site-specific pH-guided antimicrobial activity. The presented results improve our understanding on the molecular interactions between the LL-37 and surfactant-like lipids during their self-assembly, and furthermore correlate their surface charge and structural properties with their antimicrobial activity. Establishing this is of paramount importance for further development of pH-targeted delivery systems for AMPs.


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ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Complementary cryo-TEM images, DLS correlation curves and GIFT structure factor model parameters (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] ORCID Mark Gontsarik: 0000-0001-7613-5137 Anan Yaghmur: 0000-0003-1608-773X Stefan Salentinig: 0000-0002-7541-2734 Katharina Maniura-Weber: 0000-0001-7895-3563 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources The authors acknowledge the Swiss National Science Foundation (Project 200021_169513) and the Novartis Foundation for Medical-Biological Research, both held by S.S. for funding this study. Notes


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The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors thank Ana Diaz for the technical support at the cSAXS beamline (PSI, Willingen, Switzerland) and Olga Shebanova at the I22 SAXS beamline (Diamond Light Source, Oxfordshire, UK). They are grateful to Tillmann Pape for the technical assistance with cryoTEM imaging.

ABBREVIATIONS AMP, antimicrobial peptide; OA, oleic acid; GMO, glycerol monooleate; SAXS, small angle Xray scattering; cryo-TEM, cryogenic transmission electron microscopy; DLS, dynamic light scattering; PDI, polydispersity index; GIFT, generalized indirect Fourier transformation; p(r), pair-distance distribution function; PBS, phosphate buffer saline; CPP, critical packing parameter; Dapp, apparent diffusion coefficient; CFU, colony forming units; BHI, brain-heart infusion; PC agar, plate count agar;

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TABLE OF CONTENTS FIGURE


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From Structure to Function: pH-Switchable Antimicrobial Nano-Self

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