Antimicrobial Peptide-Driven Colloidal Transformations in Liquid

Aug 19, 2016 - Designing efficient colloidal systems for the delivery of membrane active antimicrobial peptides requires in-depth understanding of the...
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Antimicrobial Peptide-Driven Colloidal Transformations in LiquidCrystalline Nanocarriers Mark Gontsarik,†,‡,§ Matthias T. Buhmann,†,§ Anan Yaghmur,‡ Qun Ren,† Katharina Maniura-Weber,† and Stefan Salentinig*,† †

Laboratory for Biointerfaces, Department Materials meet Life, Empa, Swiss Federal Laboratories for Materials Science and Technology, Lerchenfeldstrasse 5, 9014 St. Gallen, Switzerland ‡ Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark S Supporting Information *

ABSTRACT: Designing efficient colloidal systems for the delivery of membrane active antimicrobial peptides requires in-depth understanding of their structural and morphological characteristics. Using dispersions of inverted type bicontinuous cubic phase (cubosomes), we examine the effect of integrating the amphiphilic peptide LL-37 at different concentrations on the self-assembled structure and evaluate its bactericidal ability against Escherichia coli. Small-angle X-ray scattering, dynamic light scattering, and cryogenic transmission electron microscopy show that LL-37 integrates into the bicontinuous cubic structure, inducing colloidal transformations to sponge and lamellar phases and micelles in a concentration-dependent manner. These investigations, together with in vitro evaluation studies using a clinically relevant bacterial strain, established the composition−nanostructure−activity relationship that can guide the design of new nanocarriers for antimicrobial peptides and may provide essential knowledge on the mechanisms underlying the bacterial membrane disruption with peptide-loaded nanostructures.

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interest in loading LL-37 into GMO-based nanocarriers is its antibacterial activity against clinically relevant bacterial strains.21 Owing to its amphiphilic nature, LL-37 targets the bacterial membrane, presumably integrating into its structure and eventually promoting its destabilization.22,23 Similar to other membrane-active AMPs, LL-37 provides a much needed alternative to conventional antibiotics and is of particular interest as it is thought to provoke less resistances.24 To achieve efficient oral delivery and long circulation times, a carrier is desired that protects this AMP from proteolytic enzymes originating from exposure to the gastrointestinal tract, plasma, or bacteria itself.25−28 We believe that this is the first report of peptide-induced colloidal transformations from a dispersed inverse bicontinuous cubic phase, so-called cubosomes, to vesicles and direct micelles, simply by the addition of LL-37 to GMO-based cubosomes. Small angle X-ray scattering (SAXS), dynamic light scattering (DLS), and cryogenic transmission electron microscopy (cryo-TEM) were used to characterize and visualize the colloidal structures. In vitro studies using a clinically relevant bacterial strain established the composition−nanostructure− activity relationship. This combination of biophysical and in vitro investigations offers a good framework for understanding

ntimicrobial peptides (AMPs) represent a good alternative to conventional antibiotics.1 However, their solubility in water is generally low and their antimicrobial activity and stability due to chemical degradation in biological systems are poor.2 Emulsified liquid crystals with their ability to solubilize hydrophobic, hydrophilic, and amphiphilic molecules have been of interest recently in the pharmaceutical delivery field.3−5 The formation of such highly organized systems was recently also discovered during the lipase-catalyzed digestion of milk fat and linked to their function as carriers for poorly water-soluble molecules.6 Such colloids offer high solubilization capacity and potential for controlled and targeted release of the encapsulated bioactives through control of the internal nanostructure, and they can protect their cargo from chemical degradation.4,7−16 The generation of these colloidal structures is generally achieved by the packing of carefully selected lipids and surfactants, and can generally be described by the critical packing parameter (CPP) model.17−20 This approach also allows the design of stimuli-responsive delivery systems, where a phase change can be achieved with an external stimulus, inducing the release of the loaded active component.2,14 Here we report on the formation and characterization of colloidal structures with self-assembled interiors based on mixtures of glycerol monooleate (GMO) and the amphiphilic α-helical antimicrobial peptide LL-37, where the peptide is actively incorporated into the interfacial lipid−water film and therefore participates in the self-assembly process. Of particular © 2016 American Chemical Society

Received: July 22, 2016 Accepted: August 19, 2016 Published: August 19, 2016 3482

DOI: 10.1021/acs.jpclett.6b01622 J. Phys. Chem. Lett. 2016, 7, 3482−3486

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The Journal of Physical Chemistry Letters

Im3m phase. With further increase in LL-37 content to GMO:LL-37 weight ratios of 90:10, 80:20, and 60:40, the reflections of the Im3m phase gradually disappear promoting structural transitions to the L3 phase and multilamellar vesicles with d spacing of ∼6 nm (Figure S1). For 50:50 w/w GMO:LL-37, the scattering pattern is dominated by small structures such as normal micelles or bicelles (Figure 1). This transition from lamellar structures to micelles follows the observations on amphiphilic peptide/phospholipid interactions reported in previous studies.33,34 The transition from cubosomes to small nanostructures with increasing LL-37 content is associated with disappearance of the low-q upturn in the I(q), which is typically observed for particles with dimensions above the resolution limit of the SAXS setup. However, a slight slope in the low-q range can still be observed for this sample, which may be attributed to the coexistence of a small fraction of larger vesicular structures, as confirmed by cryo-TEM (Figure 2). The colloidal transformations from the turbid nanostructured dispersions to transparent solutions of micelles or small vesicles are also observed by visual inspection of the samples (Figure 1). Analysis of the scattering data for the 50:50 w/w GMO:LL37 system with the generalized indirect Fourier transformation (GIFT) method showed charged core−shell type structures with an overall dimension of ∼12 nm (Table S1 and Figure S2).35 The cationic peptide, associated with the hydrophilic glycerol groups of the GMO at the interface, is thought to be responsible for the higher excess electron density in the shell surrounding the hydrophobic core, mainly formed by the fatty acid tails of GMO molecules. This accumulation of the peptide is further supported by ζ-potential measurements showing an increase in the nanoparticle charge from −11 to +12 mV with augmenting LL-37 concentrations (Figure S3). Complementary results from cryo-TEM observations were in agreement with SAXS data analysis. At 95:5 w/w GMO:LL-37, cubosomes and sponge-like nanoparticles are dominating (Figure 2). Coexisting vesicles of various size and shapes are also visible. At 90:10 GMO:LL-37, sponge-like nanoparticles and vesicles, and at 50:50 GMO:LL-37, micelles or bicelles and lamellar structures are dominating the image. Additional DLS measurements show the effect of LL-37 integration on the size characteristics of GMO-based cubosomes: The hydrodynamic radius (RH) was ∼187 nm (polydispersity index = 0.17) in the absence of LL-37 and ∼173 nm (polydispersity index = 0.22) at GMO:LL-37 = 50:50. The

LL-37-triggered phase transitions and their role in modulating the antibacterial activity, and it provides the basis for further studies on the mechanisms underlying the bacterial membrane disruption with peptide-loaded nanostructures. Figure 1 shows the SAXS pattern for a GMO dispersion stabilized by the polymeric stabilizer F127 in absence of LL-37.

Figure 1. SAXS patterns of the GMO-based samples with varying GMO:LL-37 w/w ratios at 25 °C. The first three peaks for the bicontinuous cubic Im3m phase are identified with their Miller indices. The composition-dependent change in lattice dimensions and transition to sponge and lamellar structures as well as normal micelles and small vesicles at highest LL-37, content are shown. The pictures on the right show that the samples are (a) turbid at low LL-37 content (cubosomes) and (b) transparent at high LL-37 content (vesicles and micelles).

The first three Bragg reflections of an inverse bicontinuous cubic Im3m phase with a lattice constant a ≈ 14 nm were detected, in good agreement with previous reports.29−31 In the presence of small concentrations of LL-37 the internal nanostructure is still of Im3m symmetry, but it becomes larger as its characteristic peaks shift to lower q values with a ≈ 15 nm. An additional, diffuse Bragg peak occurs at q ≈ 0.3 nm−1 for 95:5 w/w GMO:LL-37, indicating most likely a coexistence of Im3m and sponge (L3) phase.32 The significant impact of positively charged LL-37 on the nanoparticles’ interiors is attributed to its transfer from the surrounding continuous aqueous medium and integration into the lipid bilayers of the

Figure 2. Cryo-TEM micrographs of nanoparticles with GMO:LL-37 w/w ratios of (a) 95:5, (b) 90:10, and (c) 50:50. The highly ordered internal structure of cubosomes, denoted as ‘1’, is absent at higher LL-37 content. Sponge-like nanoparticles (‘2’), micelles or bicelles (‘3’) with vesicles, and multilamellar structures (‘4’) appear with increasing LL-37 content. The large dark areas represent the carbon grid. The corresponding SAXS scattering curves are presented in Figure 1 and Figure S1. 3483

DOI: 10.1021/acs.jpclett.6b01622 J. Phys. Chem. Lett. 2016, 7, 3482−3486

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The Journal of Physical Chemistry Letters corresponding DLS correlation functions show relatively monodisperse nanoparticles (Figure S3). The larger hydrodynamic radius for nanoparticles in the 50:50 GMO:LL-37 sample compared with dimensions from the SAXS analysis originates from the strong scattering contribution of few larger structures, also visible in the cryo-TEM images. The near 4-fold decrease in scattering intensity at 90° scattering angle was observed for these samples as a result of an increase in the concentration of relatively small particles at the cost of larger ones, confirming the observations from SAXS and cryo-TEM. The SAXS results and cryo-TEM images indicate that the positively charged peptide preferentially localizes in the curved bilayers of the cubosomes, and this integration induces significant alterations in the structural and morphological properties. Clearly, LL-37 induces an enlargement of the bicontinuous cubic structure and then enhances structural transitions to vesicular structures and disjoint micelles in a concentration-dependent manner (Table S2). Steric and electrostatic interactions between the cationic peptide molecules at the lipid water interface, with the associated increase in the CPP, are thought to be responsible for these colloidal transformations. It is plausible that this LL-37-triggered transition agrees with the theoretical description of the transformations between folded bilayers and micelles via the formation and growth of punctures, followed by membrane tearing.36 The distinct colloidal structures in the binary GMO:LL-37 mixture allow establishing the nanostructure− activity relationship. The reported transition from cubosomes to vesicles and even micelles with increasing the peptide concentration can be tuned back to cubosomes or even further to hexosomes or emulsified microemulsions by varying the lipid composition.18 The possible influence of nanostructural properties on the antibacterial activity of LL-37 was investigated in broth microdilution assays. Selected LL-37 nanocarriers were studied in Escherichia coli (Figure 3). Cubosomes prepared at binary GMO:LL-37 mixtures of 99:1 and 95:5 showed no significant antibacterial activity as compared with LL-37-free cubosomes. In these samples, the accommodation of LL-37 in the hydrophilic nanochannels of the internal bicontinuous cubic phase might inhibit the direct contact of the peptide with the bacterial membrane. Loaded into micelles and small vesicles, at a GMO:LL-37 w/ w ratio of 50:50, LL-37 showed significant antibacterial effects: a more rapid elimination of bacteria and more sustainability than free LL-37 (Figure 3). These results suggest that the micelles and small vesicular aggregates act as effective shuttles transporting higher loads of peptide to the inner bacterial membranes. The positive charge of the LL-37 loaded micelles with a ζ-potential of +12 mV (see Figure S3) may also result in attraction and accumulation of these carriers on the negatively charged bacterial membrane. This is in agreement with reports on the importance of electrostatic interaction in accumulation of cationic components at the negatively charged bacterial membrane.37,38 In conclusion, LL-37 was solubilized in the internal structure of GMO-based cubosomes. The resulting GMO:LL-37 system showed strong response to composition as the highly ordered bicontinuous cubic Im3m structure was transformed to sponge phase and lamellar and micellar structures with increasing LL37 concentration. Our results also indicate higher antibacterial effect for the LL-37-loaded micelles compared with the free peptide, suggesting improved delivery to the bacterial

Figure 3. Bactericidal activity against E. coli of different LL-37 nanoparticlulate formulations. The bacterial culture was treated with 80 μg/mL LL-37 in the form of a free solution (positive control) or as part of selected self-assembled nanostructures: The GMO:LL-37 = 50:50 micelles killed significantly more bacteria after 30 and 60 min compared with free LL-37 (student’s t test p < 0.05). LL-37 formulated as cubosomes (at 95:5) had no significant effect (ANOVA, p < 0.05). Unloaded cubosomes had no effect on cell population and were used as negative control. Additional measurements and controls are presented in Figure S5.

membrane. The observed results are interesting for the design of controlled and targeted delivery systems for amphiphilic antimicrobial peptides such as LL-37.

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EXPERIMENTAL METHODS Detailed information on materials and methods is presented in the Supporting Information. In short, GMO was dispersed in PBS pH 6.5, with F127 as stabilizer, using a tip sonicator and was allowed to equilibrate for at least 12 h. Different weight fractions of GMO were replaced by LL-37, keeping the total weight of particle constituents (GMO + LL-37) at 5% w/w. Nanoparticles with different GMO:LL-37 ratios were investigated using small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS), and an in vitro broth microdilution study on E. coli.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.6b01622. Detailed description of experimental methods and materials, additional SAXS data with model-independent analysis, results from dynamic light scattering, and ζpotential measurements as well as further data on antibacterial activity against E. coli. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +41 58 765 7202. E-mail: [email protected]. Author Contributions §

M.G. and M.T.B. contributed equally to this work.

Notes

The authors declare no competing financial interest. 3484

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ACKNOWLEDGMENTS We acknowledge Empa for funding this project. The scattering studies in this manuscript were conducted at the Austrian SAXS beamline (Elettra synchrotron station, Trieste, Italy). We thank Heinz Amenitsch for the technical support and fruitful discussion. A.Y. further acknowledges travel support from the Danish Natural Sciences Research Council (DanScatt). We are grateful to Klaus Qvotrup and Diana Intan Mat Azmi for the technical assistance with cryo-TEM imaging.

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DOI: 10.1021/acs.jpclett.6b01622 J. Phys. Chem. Lett. 2016, 7, 3482−3486