Lipid-Based Liquid Crystals As Carriers for Antimicrobial Peptides

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Lipid-based liquid crystals as carriers for antimicrobial peptides: phase behavior and antimicrobial effect Lukas Boge, Helena Bysell, Lovisa Ringstad, David Wennman, Anita Umerska, Viviane Cassisa, Jonny Eriksson, Marie-Laure Joly-Guillou, Katarina Edwards, and Martin Andersson Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00338 • Publication Date (Web): 31 Mar 2016 Downloaded from http://pubs.acs.org on April 1, 2016

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Lipid-based liquid crystals as carriers for antimicrobial peptides: phase behavior and antimicrobial effect Lukas Boge*1,6, Helena Bysell1, Lovisa Ringstad1, David Wennman2, Anita Umerska3, Viviane Cassisa4, Jonny Eriksson5, Marie-Laure Joly-Guillou4, Katarina Edwards5, Martin Andersson6 1

SP Technical Research Institute of Sweden, Drottning Kristinas väg 45 Box 5607 Stockholm SE 11486, +46105166078 [email protected], 2 SP Process Development, Forskargatan 18 Box 36 Södertälje SE 15121, 3 Inserm U1066 University of Angers, 4 rue Larrey Cedex 9 Angers FR 49933, 4 Laboratoire de Bactériologie-hygiène, CHU Angers 4 rue Larrey Angers FR 49000, 5 Uppsala University, Department of Chemistry – BMC Husargatan 3 Box 579 Uppsala SE-75123, 6 Chalmers University of Technology, Department of Chemical and Chemical Engineering, Applied Chemistry Kemigården 4 Göteborg SE-41296

Abstract The number of antibiotic-resistant bacteria is increasing worldwide and the demand for novel antimicrobials is constantly growing. Antimicrobial peptides (AMPs) could be an important part of future treatment strategies of various bacterial infection diseases. However, AMPs have relatively low stability due to proteolytic and chemical degradation. As a consequence, carrier systems protecting the AMPs are highly needed for achieving efficient treatments. In addition, the carrier system also needs to administrate the peptide in a controlled manner to match the therapeutic dose window. In this work, lyotropic liquid crystalline (LC) structures consisting of cubic glycerol monooleate/water and hexagonal glycerol monooleate/oleic acid/water have been examined as carriers for AMPs. These LC structures have capability of solubilizing both hydrophilic and hydrophobic substances as well as being biocompatible and biodegradable. Both bulk gels and discrete dispersed structures, i.e. cubosomes and hexosomes have been studied. Three AMPs named AP114, DPK-060 and LL-37 have been investigated with respect to phase stability of the LC structures and antimicrobial effect. Characterization of the LC structures was performed using smallangle x-ray scattering (SAXS), dynamic light scattering, ζ-potential, and cryogenic transmission electron microscopy (Cryo-TEM) and peptide loading efficacy by ultra-performance liquid chromatography. The antimicrobial effect of the LCNPs was investigated in-vitro using minimum inhibitory concentration (MIC) and time-kill assay. The most hydrophobic peptide (AP114) was shown to induce an increase in negative curvature of the cubic LC system. The most polar peptide (DPK-060) induced a decrease in negative curvature while LL-37 did not change the LC phase at all. The hexagonal LC phase was not affected by any of the AMPs. Moreover, peptides AP114 and DPK-060 loaded cubosomes showed preserved antimicrobial activity, whereas LL-37 loaded particles displayed a loss in its broad spectrum bactericidal properties. AMP loaded hexosomes showed a reduction in antimicrobial activity.

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Introduction The number of antibiotic resistant bacteria is constantly increasing and has become a vast problem in the global health sector. Antimicrobial peptides (AMPs) are present in all organisms as part of the innate defense system. These peptides are promising therapeutics to treat various infectious diseases, due to their fast and non-specific mechanism of action and because bacteria are less prone to develop high-level resistance towards these antimicrobials [1, 2]. AMPs are generally constituted of fewer than 50 amino acids, have a positive net-charge and often being amphiphilic [2]. The main challenge for clinical translation of AMPs has previously been the lack of chemical and proteolytic stability, but today these issues can be overcome by clever modifications and formulation strategies, such as incorporating them into well designed drug delivery vehicles [3]. Different types of delivery systems have been investigated for peptide delivery, such as a variety of lipid-based formulations [4, 5]. Lyotropic liquid crystalline (LC) structures of polar lipids have potential applications as carriers and delivery systems in various pharmaceutical applications. This is due to their ability to solubilize and encapsulate both hydrophilic and hydrophobic substances. An important feature of these LC systems is that they can coexist with excess of water which enables fragmentation of bulk gels into LC nanoparticles (LCNPs) in the presence of a suitable stabilizer [6, 7]. Polar peptides and proteins can be incorporated in the lipid self-assembled structures and thus being less subjected to chemical and proteolytic degradation [8-11]. LCNPs has been investigated as carriers for the delivery of several peptides and proteins; insulin [12], ovalbumin [13-15], somatostatin [16] and cyclosporine A [17, 18] are a few examples. These studies report encouraging results in terms of observed blood glucose values (insulin, in-vivo, rat), sustained release properties (ovalbumin, in-vivo, rat), increased half-life time (somatostatin, in-vivo, rat) and increased peptide skin penetration (cyclosporine A, in-vivo, mice). One of the most extensively studied LC system in drug delivery is the glycerol monooleate (GMO)/water system [19]. Depending on the nature of introduced guest molecules in such LC systems, e.g. the polarity and molecular structure, the molecules will be located either in the water or lipophilic domains of the system [20]. It has been shown that addition of lipophilic compounds to the GMO/water LC cubic system induce transition to a reverse hexagonal phase (increased negative curvature), while molecules having a pronounced amphiphilic character can induce transition to a lamellar LC phase (decreased negative curvature) [19, 21, 22]. AMPs comprising differences in amphiphilicity could be prone to induce phase transitions, as described above, when incorporated in such systems. LC phase transitions have previously been shown for a variety of protein-LCNP systems [23-29]. Little is today known about LCNPs as drug delivery vehicles for AMPs and whether the antimicrobial effect can be preserved or even improved. Only a few antimicrobial peptides have so far been formulated in LC gels or colloidal lipid formulations. One example is the antimicrobial peptide melittin, which was successfully incorporated in a lipid-disk formulation [30]. The formulation showed preserved antimicrobial effect, as compared to unformulated peptide. Also, the lipid-disk formulation protected the peptide for proteolytic degradation and demonstrated bactericidal properties upon repeatedly exposure to bacteria, indicating a sustained release of peptides from the lipid disks. Moreover, different liposomal formulations have been investigated for delivery of several AMPs [31].


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In this study the AMPs named AP114, DPK-060 and LL-37 were investigated together with LC systems as carrier. The three water soluble AMPsdisplay differences in antimicrobial selectivity, origin and physical properties. The peptide AP114 (also known as NZ2114) is an improved derivative of plectasin, a defensin peptide produced by the fungus Pseudoplectania nigrella [32, 33]. In contrast to other AMPs, acting by disrupting bacterial membranes, AP114 inhibits the membrane biosynthesis by targeting the cellular precursor Lipid II [32]. AP114 is active against Gram-positive bacteria, including Staphylococcus aureus and its methicillin resistant variety (MRSA), making it useful in treatment of pneumonia. The DPK-060 peptide is derived from the endogenous human protein kininogen and is intended for topical administration to treat a variety of infected skin conditions. Its effect has been confirmed by clinical phase II studies as treatment for infections in atopic dermatitis and acute external otitis [3]. LL-37 is the only known human peptide in the cathelicidin family, found in different cells, tissues and body fluids [34]. LL-37 also has wound-healing properties, besides its broad spectrum antibacterial activity, making it suitable for topical administration on infected wounds. The peptide is sensitive to various bacterial proteolytic enzymes, which so far has limited its therapeutic use [35]. Briefly, the purpose of this present research was to investigate how three different AMPs influenced the LC structure of a cubic (GMO/water) and hexagonal (GMO/oleic acid/water) gel and to evaluate the antimicrobial effect of dispersions of those, that is cubosomes and hexosomes. Characterization of the LC gels was performed using small angle x-ray scattering (SAXS). The peptide loaded LCNPs were characterized in terms of particle size, ζ-potential, structure (SAXS and cryogenic Transmission Electron Spectroscopy, Cryo-TEM) and peptide loading efficacy (Ultra Performance Liquid Chromatography, UPLC). The antimicrobial effect of the LCNPs was investigated in-vitro using minimum inhibitory concentration (MIC) tests.

Experimental Materials Glycerol monooleate RYLO MG 19 Pharma was purchased from Danisco A/S (Grindsted, Denmark). The composition of the sample according to the manufacturer was min. 95 % monoglycerides, max. 10 % diglycerides and max. 2 % triglycerides. The fatty acid content was minimum 88 % oleoyl (C18:1). Super refined oleic acid NF-LQ-(MH) was purchased from Croda Inc. (Snaith, UK). The triblock copolymeric stabilizer poly(ethylene oxide)(PEO)-poly(propylene oxide)(PPO)-poly(ethylene oxide)(PEO) with trade name Lutrol F127 was obtained from BASF (Lundwigshafen, Germany) and had an approximate formula of PEO101PPO56PEO101 and an average molecular weight of 12600 g/mol. AP114 (purity 99.1 %) was provided by Adenium Biotech ApS (Copenhagen, Denmark), DPK-060 (purity 98.5 %) was synthesized by Bachem AG (Bubendorf, Swizerland) and provided by Pergamum AB (Stockholm, Sweden) and LL-37 (purity 94.7 %) was synthesized and provided by PolyPeptide Laboratories (Limhamn, Sweden). The structure and properties of the peptides are summarized in Table 1. All substances were used as received.


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Table 1. Structure and properties of the AMPs. All peptides were soluble in water at the used concentrations. Amino acid (AA) color code: Black letters=neutral, Red=lipophilic, blue= positively charged, yellow=negatively charged. Peptide

AP114 DPK-060 LL-37

Sequence HGFGCNGPWNEDDLRCHNHCKSIKGYKGG YCAKGGFVCKCY-OH H-GKHKNKGKKNGKHNGWKWWW-OH HLLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLV PRTES-OH

MW

% hydrophobic AA

Net charge (pH=5.5)

4411

40

+4.6

2503

20

+8.5

4491

35

+6.3

Secondary structure

Antibacterial activity

β-sheet Gram-positive Linear, random coil Linear α-helix, random coil

Broad spectrum Broad spectrum

Sample preparation LC gels were prepared by mixing melted GMO or GMO/OA mixtures (40 °C) with a water solution containing AMP. Samples were homogenized with spatula, centrifuged at 3000 rpm for 1 hour and allowed to equilibrate at room temperature for at least 1 week prior further use. The lipid to water ratio for the LC gels prepared without AMP were chosen to obtain the cubic or hexagonal phase, so the peptide’s impact of the curvature could be investigated. LCNP dispersions were made by fragmenting 5 w % (0.5 g) of the LC gel in 9.5 g 1.0 % Lutrol F127, dissolved in 5mM acetic acid buffer pH=5.5, with a Ultra-Turrax high shear mixer (IKA T25, Staufen, Germany) operated at 15.000 rpm for 1-2 min. The coarse dispersion were ultra-sonicated using a Vibra-Cell VC 750 (Sonics and Materials Inc., Newton, USA) with a 6 mm probe operating at 40 % of its maximum power in puls mode (3 s pulses followed by 7 break) for a total time of 5 min (GMO gels) or 10 min (GMO/OA gels). The GMO/OA ratio (8:2) was chosen according to data presented by Salentinig et al. (2010), and was shown to result in LCNPs with hexagonal structure at pH=5.5 [22]. Table 2 summarizes the prepared samples. Table 2. Summary of the composition of cubic and hexagonal LC gels and LCNP dispersions that were analyzed in the study. The LCNPs were made by dispersing 5 w % LC gel in 5mM acetic acid buffer pH=5.5 containing 1 % Lutrol F127 stabilizer.

Sample code

Lipid composition GMO:OA (w %)

Cub-ref 100:0 Cub-AP-0.25 100:0 Cub-AP-0.5 100:0 Cub-AP-1.0 100:0 Cub-AP-1.5 100:0 Cub-DP-0.5 100:0 Cub-DP-1.0 100:0 Cub-DP-1.5 100:0 Cub-LL-0.5 100:0 Cub-LL-1.0 100:0 Cub-LL-1.5 100:0 Hex-ref 80:20 Hex-AP-0.5 80:20 Hex-AP-1.0 80:20 Hex-AP-1.5 80:20 Hex-DP-0.5 80:20 Hex-DP-1.0 80:20 Hex-DP-1.5 80:20 Hex-LL-0.51 80:20 Hex-LL-1.0 80:20 1 Hex-LL-1.5 80:20 1 Samples were not analyzed by SAXS.

Lipid:aqeous phase for LC gels (w %) 70:30 70:30 70:30 70:30 70:30 70:30 70:30 70:30 70:30 70:30 70:30 80:20 80:20 80:20 80:20 80:20 80:20 80:20 80:20 80:20 80:20

AMP

AP114 AP114 AP114 AP114 DPK-060 DPK-060 DPK-060 LL-37 LL-37 LL-37 AP114 AP114 AP114 DPK-060 DPK-060 DPK-060 LL-37 LL-37 LL-37

AMP concentration in LC gels (w %) 0 0.25 0.5 1.0 1.5 0.5 1.0 1.5 0.5 1.0 1.5 0 0.5 1.0 1.5 0.5 1.0 1.5 0.5 1.0 1.5


Final AMP concentration (µg/ml) in dispersions 0 125 250 500 750 250 500 750 250 500 750 0 250 500 750 250 500 750 250 500 750

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Particle size measurements The particle size and its distributions was measured by dynamic light scattering using a Ζetasizer Zen3600 instrument (Malvern Instruments Ltd., Worcestershire, U.K.) using disposable cuvettes. The samples were diluted to 2-2.5 mg/ml in MilliQ water prior to analysis and each sample was measured in triplicate. Refractive indices were set to 1.47 and 1.33, for lipids and water respectively, and the temperature were held at 25 °C during measurements. The software calculates the hydrodynamic radius, assuming spherical particles, using Stoke-Einstein´s relation to describe the Brownian motion of the particles.

ζ-Potential The ζ-potential of the particles was determined by measuring the electrophoretic mobility with a Ζetasizer Zen3600 (Malvern Instruments Ltd., Worcestershire, U.K.) using the Smoluchowski model. Samples were diluted in MilliQ water to a particle concentration of 2-2.5 mg/ml and analyzed in disposable measuring cells at 25 °C. Each sample was measured in triplicate.

Small angle x-ray scattering (SAXS) Synchroton SAXS measurements were performed at beamline I911-SAXS (MAX VI Laboratory, Lund, Sweden) equipped with a fast read-out pixel detector PILATUS 1M (Dectris Ltd., Baden, Switzerland) [36]. Liquid crystalline gels were mounted in a multiple sample holder made of steel and fitted between kapton sheets, whereas liquid samples were loaded into quartz capillaries (ID 1.0 mm) mounted in a steel holder. Samples were subjected to a flux of 5 ∙ 10 photons/s with a beam size of 0.3×0.3 mm2 at a wavelength of 0.91 Å. The exposure time was 100 sec for each sample. Measurements were carried out in air at 22 and at 37 °C for selected samples. Collected raw-data images were analyzed using the software Bli911-4 GUI version 4.12 and the LC mesophases were identified according to Bragg peak spacings [37].

Cryogenic Transmission Electron Microscopy (Cryo-TEM) For Cryo-TEM, specimens were prepared in a controlled environment vitrification system kept at 25 °C with humidity close to saturation to prevent evaporation from the sample during preparation. A droplet of the LCNP dispersion, diluted 5X in MilliQ, was placed onto a carbon-coated holey polymer film supported by a copper grid, gently blotted with a filter paper to form a thin liquid film (10-500 nm), and immediately plunged into liquid ethane at -180 °C for vitrification. The sample grid was then kept at liquid nitrogen temperature and transferred into a Zeiss LIBRA-120 transmission electron microscope (Carl Zeiss, Oberkochen, Germany) operated at 80kV in zero-loss bright-field mode. Digital images were recorded under low dose conditions with a TRS slow scan CCD camera system (TRS GmbH, Germany) and iTEM software (Olympus Soft Imaging Solutions GmbH, Germany). An underfocus of 1-3 µm was used in order to enhance contrast. The cryo-TEM technique has been described in detail elsewhere [38].

Ultra Performance Liquid Chromatography (UPLC) The peptide loading efficacy of AMPs in the LCNPs was quantified indirectly by separating the LCNPs from the surrounding liquid by centrifugation at 15000xg for 20 - 40 min through Amicon Ultra-0.5 filter devices (Ultracel-100K, Merck Millipore Ltd. Corc, Ireland) with a 100 kDa molecular weight cutoff. The peptide concentration in the filtrate was then quantified using a Waters Acquity UPLC system (Waters Corp., Milford Massachusetts, USA) with a UV-detector. The system was equipped with a C18-column 1.7 µm, 2.1 x 150 mm (Acquity BEH300, Waters Corp., Milford Massachusetts,


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USA) operated at 40 °C. The mobile phases used were acetonitrile/water 10/90 by volume plus 0.03 % trifluoroacetic acid (phase A) and acetonitrile/water 90/10 by volume plus 0.03 % trifluoroacetic acid (phase B) at a flow rate of 500 µl/min. A gradient where the mobile phase composition was changed from 100 % phase A to 39 % over a period of 3.4 min was employed. The injection volume was 5 µl and the absorbance was measured at 205 (LL-37) or 215 (AP114 and DPK-060) nm. The concentration of peptide in the filtrate was determined using a calibration curve and a correction factor compensating for adsorption to the filter. The amount peptide incorporated or adsorbed to the LCNPs could then be calculated, as % of total added peptide.

Minimum inhibitory concentration (MIC) The bacteriostatic behavior of pure peptides and peptide loaded LCNPs was studied using the broth microdilution method. The bacteria used were Staphylococcus aureus, reference strain ATCC 25923, methicillin-resistant S. aureus (MRSA) clinical strain No. 0702E0196, Pseudomonas aeruginosa, reference strain ATCC 27853 and clinical strain No. 0704C0134, Escherichia coli, reference strain ATCC 25922, and ESBL E. coli, clinical strain ATCC 9007550201, and Acinetobacter baumannii, AYE reference strain ATCC BAA-1710. Inoculum was prepared by taken 1-2 colonies directly from a Columbia agar plate supplemented with sheep blood (Oxoid, France) into 2 ml of 0.85% NaCl solution, and the density of the microorganism suspension was adjusted to approximately 3.3*108 colony forming units (CFU)/ml (S. aureus) or to approximately 1.5*108 CFU/ml (P. aeruginosa, E. coli, A. baumannii). The former suspensions were further diluted 100 times (3.3*106 CFU/ml), and the latter were diluted 10 times (1.5*107 CFU/ml) with brain heart infusion broth (BHI) (bioMérieux, France) for AP114 containing test items or 1% BHI in water for DPK-060 and LL-37 containing test items.

Serial two-fold dilutions of the tested AMP loaded LCNP samples in BHI or 1% BHI in water were prepared in order to obtain the desired concentration range. An aliquot of 100 μl of the bacterial suspension was added into each well of a sterile 96 well plate containing 100 μl of the tested sample. Positive controls containing only the medium and the bacterial suspension (growth control) and negative control wells, containing the medium and the tested sample without the bacterial suspension (sterility control), were also prepared. The plates were incubated at 37 °C for 24 hours. The MIC was defined as the lowest concentration of the sample that completely inhibited the growth of the bacteria as detected by the unaided eye. If the assessment was not possible due to turbidity of the sample, an amount of approximately 2 μl was withdrawn from each well, transferred onto a plate with Mueller Hinton agar using an multipoint inoculator (AQS Manufacturing LTD, UK) and incubated for 24 hours at 37 °C.

Time-kill assay To evaluate the bactericidal activity of peptide-loaded LCNPs, time-kill assays were performed. An inoculum of approximately 3.3*104 CFU/ml was added to a final volume of 2 ml in a polypropylene tube. The samples containing the tested LCNPs in BHI, as well as the controls were incubated at 37 °C. At each sampling time (0, 3, 6, 18 and 24 hours) an amount of 100 μl was withdrawn from each tube. Serial 100-fold dilutions were prepared in distilled water when necessary. A 100 μl aliquot of the diluted and/or undiluted sample was delivered onto the surface of the agar and allowed to be absorbed into the agar. Having incubated the agar plates for 24 hours at 37°C, the colonies were counted.


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Results Characterization of AMP loaded LC gels First, the effect of incorporating AMPs into cubic and hexagonal LC gels was studied using SAXS, and diffractograms are presented in Figure 1. Characterizations of the dispersed LC gels are shown in the next section. As can be seen, when peptides were added to the cubic gel, the LC structure was kept (LL-37), changed to another cubic symmetry (DPK-060) or gradually turned into a hexagonal phase (AP114). The LC structure of the hexagonal gels was unaltered upon incorporation of any of the AMP at the studied concentrations. The unloaded reference cubic GMO LC gel showed eight distinct Bragg reflections with spacing ratios of √6: √8: √14: √16: √20: √22: √24: √26. These reflections are characteristic for a not fully hydrated GMO phase, belonging to the body-centered cubic Ia3d space group. Calculated lattice parameters for the gels can be found in Table 3. Figure 1 (A) shows how the diffraction pattern changes upon increased concentration of AP114. At low peptide concentrations (≤0.5 w %) the negative curvature decreased, as the Pn3m cubic space group dominates, with peak spacing ratios of √2: √3: √4: √6: √8: √9√11: √14 . However, at higher AP114 concentrations the structure was gradually transformed to a hexagonal phase with characteristic spacings of 1: √3: √4 . This transition reflects an increase in negative curvature. Unit cell dimensions for the Pn3m increased slightly with increased AP114 content, whereas the dimension for the H2 phase decreased, as displayed in Table 3. DPK-060 gradually changed the structure from cubic Ia3d to cubic Pn3m (Figure 1 (C)), equivalent to a decrease in negative curvature. The lattice parameter for both the Ia3d and Pn3m phase in DPK-060 system decreased slightly with increasing peptide concentration. Moreover, the peptide LL-37 did not change the cubic Ia3d LC phase at the studied concentrations (Figure 1 (E)). However, peaks were slightly shifted to higher q-values, reflecting a decrease in lattice parameter.


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Figure 1. SAXS diffractograms of cubic GMO:water 70:30 w/w % (A, C, and E) and hexagonal GMO:OA:water 64:16:20 w/w/w % (B, D and F) LC gels containing AMPs AP114, DPK-060 and LL-37. Cubic gels with AP114 gradually changed its LC structure to hexagonal, while DPK-060 induced formation of another cubic phase; Pn3m. LL-37 did not induce phase changes in the cubic system. The hexagonal GMO/OA gels did not change LC phase upon addition of any of the peptides. Representative peak indexing for the cubic Ia3d (A), hexagonal (B) and cubic Pn3m (C) symmetries are displayed as the corresponding Miller indices [hkl]. Measurements were carried out at 22 °C.

Unlike the cubic GMO gels, the hexagonal GMO/OA gels kept their LC structure upon peptide addition. The lattice parameter for the HII phase without AMP, 48.9 Å, was increased irrespective of which AMP that was introduced. DPK-060 increased the hexagonal lattice parameter the most, up to 56.9 Å for the 1.0 % sample. The lattice parameter for the hexagonal gel loaded with 1 w % LL-37 was calculated to 54.4 Å, which is in between the lattice parameter for the AP114 and DPK-060 samples.


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Table 3. Calculated lattice parameters, a (Å), for the different space groups present in the AMP loaded cubic and hexagonal LC gels. Hydration level of cubic (GMO) LC gels was 30 w % and for hexagonal (GMO/OA) LC gels 20 w %. Samples were measured at 22 °C. Gel sample

Phase

a (Ia3d) (Å)

Ia3d 141.3/134.81 Pn3m Pn3m, H2+traces 139.5 of Im3m and Ia3d Cub-AP-1.0 H2, Im3m Cub-AP-1.5 H2 Cub-DP-0.5 Ia3d 133.7 Cub-DP-1.0 Pn3m, Ia3d 130.6 Cub-DP-1.5 Pn3m Cub-LL-0.5 Ia3d 135.0 Cub-LL-1.0 Ia3d 130.4 Cub-LL-1.5 Ia3d 131.4 Hex-ref H2 Hex-AP-0.5 H2 Hex-AP-1.0 H2 Hex-AP-1.5 H2 Hex-DP-0.5 H2 Hex-DP-1.0 H2 Hex-DP-1.5 H2 Hex-LL-1.0 H2 1 Results obtained from two different GMO-batches. Cub-ref Cub-AP-0.25 Cub-AP-0.5

a (Pn3m) (Å)

a (Im3m) (Å)

a (HII) (Å)

84.3 86.0

113.3

60.3

115.8

58.6 57.6

84.0 82.9

48.9 51.6 51.9 51.0 56.1 56.9 55.1 54.4

Characterization of AMP loaded LCNPs Experiments using high pressure homogenizer (microfluidizer) for making LCNP dispersions were initially performed, but resulted in dispersions dominated by vesicles, as revealed by cryo-TEM. In line with the cryo-TEM images, those samples did not display any LC structure (DPK-060 and LL-37) by SAXS and low peptide loading efficacy was observed (AP114 and DPK-060). Data is presented in Supporting information Table S2, Figure S1 and Figure S2. The internal LC structure of the dispersed LC-gels was also characterized with SAXS at 22 °C, and at physiological temperature (37 °C) for references and 1 w % AMP loaded LCNP samples. Diffractograms and calculated lattice parameters are displayed in Figure 2 and Table 4, respectively. For the GMO-based LCNPs (Figure 2A, C and E) the reference sample showed peak spacing ratios of √2: √4: √6 , indexed as (110), (200) and (211) reflections, distinctive for the Im3m cubic symmetry. Independently of which symmetry the undispersed cubic LC gel adopted, it contained the cubic Im3m space group when dispersed. As for the AP114 containing GMO gels, hexagonal phase was also present in the dispersions at the highest concentration studied. Coexisting Im3m and hexagonal phase were detected at 1.5 % peptide loading or at 1 % while kept at 37 °C. However, at 0.5 and 1.0 % AP114 content at 22 °C only the Im3m cubic phase was detected. The lattice parameter for the Im3m cubic phase decreased upon addition of AP114, while the dimension of the observed hexagonal phase did not change. Surprisingly, no diffraction peaks were detected for GMO sample containing 0.5 % DPK-060, Figure 2 (B). Samples of 1.0 % (22 and 37 °C) and 1.5 % peptide content showed characteristic peaks for the Im3m cubic symmetry, and the lattice parameter did only change slightly among the samples. LL-37 containing dispersions were all cubic Im3m and the lattice parameter analyzed at 22 °C did only differ slightly from the reference. For all samples analyzed at 37 °C, peaks were slightly shifted to higher q-values giving about 10 Å reductions in lattice parameter.


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Figure 2. SAXS diffractograms of GMO-based LCNPs loaded with AP114 (A), DPK-060 (C) and LL-37 (E) showing presence of cubic Im3m phase, and HII in case of AP114 at 1.5 w % and at 1.0 w % kept at 37 ° C. The GMO/OA-based LCNPs with AP114 (B), DPK-060 (D) and LL-37 (F) did all display hexagonal LC structure for all peptide concentrations studied. All samples were measured at 22 °C, except for samples loaded with 1 w % AMP, that were also analyzed at physiological relevant temperature (37 °C). The peptide loading in % reflects the initial peptide content in the undispersed LC gel. Representative peak indexing for the cubic Im3m (A) and hexagonal (B) symmetries are displayed as the corresponding Miller Indices [hkl]. All samples contained 5 w % gel dispersed in 5mM acetic acid buffer at pH=5.5 containing 1 % Lutrol F127 stabilizer.

The GMO/OA-based LCNPs (Figure 2 B, D and F) displayed presence of hexagonal structure and the trends observed in the lattice parameter for the undispersed gels, were also found for the LCNPs. The calculated hexagonal lattice parameter, in Table 4, varied a few Å from the gels. As for the cubic GMO-based dispersions, the lattice parameter of GMO/OA hexagonal LCNPs measured at 37 °C was slightly lower, compared to those obtained at 22°C.


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Table 4. Observed LC phases and their lattice parameters, a (Å), for dispersed GMO and GMO/OA LCNPs loaded with AP114, DPK-060 and LL-37. Samples contained 5 w % gel dispersed in 5mM acetic acid buffer at pH=5.5 containing 1 % Lutrol F127 stabilizer. Dispersed gel Cub-ref Cub-ref Cub-AP-0.5 Cub-AP-1.0 Cub-AP-1.0 Cub-AP-1.5 Cub-DP-0.5 Cub-DP-1.0 Cub-DP-1.0 Cub-DP-1.5 Cub-LL-0.5 Cub-LL-1.0 Cub-LL-1.0 Cub-LL-1.5 Hex-ref Hex -ref Hex -AP-0.5 Hex -AP-1.0 Hex -AP-1.0 Hex -AP-1.5 Hex -DP-0.5 Hex -DP-1.0 Hex -DP-1.0 Hex -DP-1.5 Hex -LL-1.0 Hex -LL-1.0

T (°C) 22 37 22 22 37 22 22 22 37 22 22 22 37 22 22 37 22 22 37 22 22 22 37 22 22 37

Phase Im3m Im3m Im3m Im3m Im3m, H2 Im3m, H2 No peaks Im3m Im3m Im3m Im3m Im3m Im3m Im3m H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2 H2

a (Im3m) (Å) 135.0 126.6 132.8 125.5 114.2 116.4

a (H2) (Å)

59.5 60.1

137.9 128.6 137.5 138.9 134.6 125.3 133.7 52.6 51.1 53.8 52.4 50.9 53.5 55.5 55.9 54.6 56.7 55.8 53.8

Furthermore, the cubic and hexagonal LCNP dispersions were characterized in terms of particle size, ζ-potential and peptide loading efficacy, and the results are illustrated in Figure 3. For the cubic LCNPs the mean particle size was always in the range of 100-130 nm with a narrow particle size distribution (PDI16 >16 -

UF 4 4 8 16 8 4-8 4-8

GMO 2-4 2-4 16 8-16 2-4 2-4 16

GMO/OA >16 >16 >16 >16 >16 >16 >16

LL-37 UF 8-16 8-16 8-16 8-16 16 16 16

GMO 64 >16 >16 8 16 >16 >16

GMO/OA >16 >16 >16 >16 >16 >16 >16

Time-kill assays were performed in order to study the bactericidal properties of the formulations over a prolonged time period, on a selection of bacterial strains according to the MIC results. The hexagonal GMO/OA LCNPs were not tested because of their high MIC-values. Data is presented in Figure 5. AP114 loaded GMO-based LCNPs were tested on S. aureus (A) and MRSA (B) and showed a similar effect as for the pure peptide (less than a 2 Log difference). DPK-060 loaded LCNPs were tested at two different concentrations, 4 and 8 µg/ml peptide, on S. aureus (C) and MRSA (D). The antimicrobial effect of the DPK-060 formulation did not differ significantly from the unformulated reference, except for the 4 µg/ml sample tested on S. aureus showing a loss in bacterial killing after 6 hours incubation time (Figure 5 C). LCNPs loaded with DPK-060 and LL-37 were also tested against E. coli and again the effect was similar to unformulated peptide. Positive controls performed as anticipated and the LCNPs did not inhibit the growth of bacteria on their own (Control and Cub-ref curves).


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Figure 5. Time-kill assay curves for GMO-based LCNPs loaded with AP114 (A, B), DPK-060 (C-E) and LL-37 (F) on a selection of bacterial strains. The bactericidal properties of the formulations (diluted to the chosen start concentration) were found to be very similar to the effect of pure peptide. Each data point is represented by mean ± standard deviation (n=3).


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Discussion In this study it was found that the cubic GMO/water gel was more sensitive to peptide additions, compared to the hexagonal GMO/OA systems, which always kept their LC structure. The nature of the peptides (hydrophobicity and net charge) strongly influenced the changes in curvature of the cubic gels. The antimicrobial effect of the cubic GMO LCNPs were in most cases comparable to unformulated peptide, while the effect for peptides loaded in GMO/OA LCNPs was drastically decreased. As expected, the unloaded reference cubic LC gel belonged to the Ia3d space group, in line with phase diagrams studies found in literature at a hydration level of 30 % [39, 40]. When the AP114 peptide was added to the system, having the highest percentage of hydrophobic residues and lowest net charge among the tested AMPs, the structure gradually turned into the hexagonal phase. Hence, AP114 is suggested to interact strongly with the hydrophobic parts of the GMO membranes, thus increasing the critical packing parameter (CPP), and in turn increasing the negative curvature. Interestingly, the cubic Pn3m phase was detected at lower peptide additions, representing a decrease in negative curvature. Transitions from Ia3d to Pn3m, and also further to HII, has previously been observed for lipophilic molecules or amphiphilic peptides added to GMO based systems [41]. As seen for AP114 at low concentrations, DPK-060 changed the structure from cubic Ia3d to cubic Pn3m above 0.5 % peptide addition. This is probably a result of DPK-060 being the most hydrophilic peptide among the examined AMPs with highest charge. DPK-060 interacts strongly with the hydrophilic head groups of GMO, resulting in a decreased CPP and in turn the negative curvature. LL-37 did not change its cubic Ia3d structure, indicating that the peptide is mostly located in the water domains of the structure or is penetrating the bilayer interfaces without significantly changing the curvature. The lattice parameter of the Ia3d phase decreased upon addition of all the investigated AMPs, at low concentrations. The change was about 10 Å for samples containing 1.0 and 1.5 w % LL-37. The difference is not likely only due to the slight decrease in water content (some water is replaced by peptide in the aqueous phase). A 1 % decrease in water content from 30 w % gives about 2 Å decrease of the Ia3d lattice parameter (extrapolation of data found in literature [39]) and one can thus expect that LL-37 makes the packing of molecules more efficient by replacing part of the GMO molecules. An interesting observation for the dispersed cubic GMO gel loaded with 1 % AP114 is the formation of a hexagonal phase upon heating at 37 °C (Table 4). An increase in temperature is known to result in dehydration of the polar head groups of nonionic amphiphiles, thus increasing the CPP and in turn the negative curvature towards hexagonal [42]. The same reasoning could be adapted to explain the decrease in lattice parameter for LCNPs kept at 37 °C. The observed increase in lattice parameter for the hexagonal GMO/OA peptide loaded gels indicates a swelling of the structure. The most hydrophobic peptide, AP114, showed the smallest increase in lattice parameter. The reason for this could be that the peptide is mostly located in the lipophilic parts of the structure, thus increasing the bulkiness and length of the hydrophobic tails, without changing the radius of the water channels. The greatest swelling was observed for DPK-060. Due to its high positive charge and hydrophilicity, DPK-060 is most prone to interact with the polar head groups and water domains in the hexagonal structure, thus swelling the cylinders diameter resulting in an increased lattice parameter. Also, electrostatic repulsion between the positive charges of the AMPs could explain the increase in lattice parameter.


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The increase in ζ-potential upon peptide incorporation is probably due to localization of the cationic AMPs at the particle’s surface. This effect has previously been observed when the positively charged peptide hormone somatostatin was adsorbed onto negatively charged LCNPs [16]. The adsorbed AMPs are most likely in equilibrium with the free peptides present in the surrounding solution. The surface and/or structure of the GMO particles loaded with DPK-060 might already been saturated at 0.5 % peptide loading, resulting in decreased loading efficacy and less increase in ζ-potential at higher concentrations, compared to the trends for AP114 and LL-37 loaded LCNPs. This indicates that the ζ-potential is not only reflecting the loading efficacy, but rather also the increase of nonencapsulated peptide. Our loading efficacies for the cubic GMO systems is comparable to the encapsulation of ovalbumin 47-53 % and cyclosporine A 86-92 % measured previously for similar carriers [13, 18]. Cryo-TEM images of both cubic and hexagonal dispersions displayed a fraction of dense particles with internal structure, in line with observed diffraction peaks by SAXS. Also, particle sizes were in agreement with DLS measurements. From Figure 4 (A) the repeating distance in the cubosome particles was measured to approximately 90-95 Å. This observation is in agreement with the calculated spacing from the (110) reflection in the SAXS-data, 135/√2 =95.5 Å. A large fraction of vesicles has previously been shown to be present in GMO/F127-based dispersions prepared by microfluidization or sonication [17, 43, 44]. The presence of bilayer vesicles has been proposed to be required during the cubosomes formation process, which appears to be slow at ambient temperatures [45]. One way of drastically increase the fraction of cubosomes is to subject the sample to a heat treatment cycle in an autoclave [43]. However, this harsh treatment was tested for microfluidized samples and resulted in phase separation or dispersions that only contained large liposomes and degraded AMP (data not shown). Compared to the GMO-based dispersions, the hexagonal GMO/OA particles appeared more spherical shaped, displayed a more fuzzy internal structure (Figure 4E-H) and did not contain any lamellar structures attached to the particle outer surfaces. These observations are in line with cryo-TEM investigations of dispersed hexagonal phases found elsewhere [17, 46-48]. DPK-060 formulated in GMO-based LCNPs appeared to be particularly effective in inhibiting growth of E. coli, compared to unformulated peptide. This indicates a synergetic effect of encapsulating DPK060 in cubic LCNPs, where not only the free (non-bounded) peptide could explain the observed antibacterial effect. The fact that the peptide loaded GMO-based LCNPs performed as well as the unformulated reference indicates that there might be a burst release of peptide from the particles. A burst release of lipophilic compounds from cubosomes has previously been shown [49], and could also be the case for the AMPs. The decrease in antimicrobial activity for the peptide loaded GMO/OA LCNPs may be explained by that peptides tend to like staying in the hexagonal environment (no phase change observed by SAXS) resulting in high peptide loading efficacy (>94 %) and a slow release from these particles. Our MIC and time-kill assay data suggest that the release from the hexagonal particles is very low during the first 24 hours. The coexistence of vesicles and cubosome and hexosome particles, inevitably bring about the question of which colloidal carrier that is encapsulating and delivering the AMPs. The observed lower peptide loading in pure vesicles (microfluidized samples) and the fact that peptides did change the LC structure (clear in the case of AP114), indicates that the peptides are immobilized in the LC structure of the cubosomes. In case of the dispersed hexagonal systems, the antibacterial effect was


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found poor, even though the peptide loading efficacy was high and the fraction of vesicles was comparable among the cubic and hexagonal systems. If the AMPs were mainly incorporated and delivered by vesicles, one would have expected that the hexagonal GMO/OA-based LCNPs should have given rise to an apparent bacterial killing effect in the MIC-tests, which were not the case. Hence, it is believed that the cubosomes and hexosomes, and not the vesicles, are the major delivery vehicles for the AMPs.

Conclusion In this study, we have demonstrated that AMPs could successfully be loaded in cubic and hexagonal LC gels and in LCNPs with at high loading efficacies. Importantly, the cubic AMP-loaded LCNPs showed promising antimicrobial activity. Moreover, it was demonstrated that the AMPs strongly influenced the LC structure of the cubic GMO/water system. Peptide net charge and degree of hydrophobicity are important factors affecting the curvature. The hexagonal GMO/OA/water system was found to be more robust upon addition of any of the AMPs, and did not change LC structure. Why peptides formulated in cubic LCNPs shows maintained antimicrobial activity, while not if formulated in hexagonal LCNPs, is not fully understood, and needs to be investigated further. Important aspects to investigate are peptide release profiles, possible release triggers from the LCNPs and interaction with bacterial membranes. Such information would provide more details about the performance in the MIC and time-kill assays. Moreover, the chemical and proteolytic stability of the AMPs formulated in LCNPs is of great interest to examine in the future.

Acknowledgement This research performed in this study was funded by the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement no 604182 within the FORMAMP project. The MAX IV laboratory is acknowledged for beam time at x-ray synchrotron beamline I911-SAXS. We would also like to thank Szymon Sollami-Delekta, SP Technical Research Institute of Sweden, (Stockholm, Sweden) for fine tuning the preparation protocol for hexosomes and Anand Kumar Rajasekharan, Chalmers University of Technology (Gothenburg, Sweden) for running part of the SAXS samples.

Supporting information. Additional tables and figures.


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Table of content

Key words: antimicrobial peptide, peptide, liquid crystal, glycerol monooleate, cubosome, hexosome


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Figure 1. SAXS diffractograms of cubic GMO:water 70:30 w/w % (A, C, and E) and hexagonal GMO:OA:water 64:16:20 w/w/w % (B, D and F) LC gels containing AMPs AP114, DPK-060 and LL-37. Cubic gels with AP114 gradually changed its LC structure to hexagonal, while DPK-060 induced formation of another cubic phase; Pn3m. LL-37 did not induce phase changes in the cubic system. The hexagonal GMO/OA gels did not change LC phase upon addition of any of the peptides. Representative peak indexing for the cubic Ia3d (A), hexagonal (B) and cubic Pn3m (C) symmetries are displayed as the corresponding Miller indices {hkl}. Measurements were carried out at 22 °C. 226x255mm (150 x 150 DPI)


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Figure 2. SAXS diffractograms of GMO-based LCNPs loaded with AP114 (A), DPK-060 (C) and LL-37 (E) showing presence of cubic Im3m phase, and HII in case of AP114 at 1.5 w % and at 1.0 w % kept at 37 ° C. The GMO/OA-based LCNPs with AP114 (B), DPK-060 (D) and LL-37 (F) did all display hexagonal LC structure for all peptide concentrations studied. All samples were measured at 22 °C, except for samples loaded with 1 w % AMP, that were also analyzed at physiological relevant temperature (37 °C). The peptide loading in % reflects the initial peptide content in the undispersed LC gel. Representative peak indexing for the cubic Im3m (A) and hexagonal (B) symmetries are displayed as the corresponding Miller Indices {hkl}. All samples contained 5 w % gel dispersed in 5mM acetic acid buffer at pH=5.5 containing 1 % Lutrol F127 stabilizer. 222x249mm (150 x 150 DPI)


Langmuir

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Figure 3. Peptide loading efficacy plotted as function of ζ-potential, for dispersion of cubic (A) and hexagonal (B) LC gels. The diameter of the bubbles is proportional against its mean particle size. The cubic reference equals 127 nm and the hexagonal 159 nm (unfilled circles). Numbers inside circles represent initial peptide loading (w %) in dispersed gels. Detailed data can be found in Supporting information Table S1. 206x91mm (150 x 150 DPI)


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Langmuir

Figure 4. Cryo-TEM images of dispersed GMO (A-D) and GMO/OA (E-H) LCNPs loaded with AP114, DPK-060 and LL-37. 1 w % peptide was initially loaded in the LC gel prior dispersion, resulting in a concentration of 500 µg/ml in the formulations. Scale bar equals 100 nm. 251x129mm (150 x 150 DPI)


Langmuir

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Figure 5. Time-kill assay curves for GMO-based LCNPs loaded with AP114 (A, B), DPK-060 (C-E) and LL-37 (F) on a selection of bacterial strains. The bactericidal properties of the formulations (diluted to the chosen start concentration) were found to be very similar to the effect of pure peptide. Each data point is represented by mean ± standard deviation (n=3). 203x272mm (150 x 150 DPI)


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Langmuir

TOC graphics. Key words: antimicrobial peptide, peptide, liquid crystal, glycerol monooleate, cubosome, hexosome 99x51mm (150 x 150 DPI)


Lipid-Based Liquid Crystals As Carriers for Antimicrobial Peptides

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