Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC
Biological and Medical Applications of Materials and Interfaces
Peptide-Loaded Cubosomes Functioning as an Antimicrobial Unit Against Escherichia coli Lukas Boge, Kathryn Louise Browning, Randi Nordstrom, Mario Campana, Liv S.E. Damgaard, Josefin Seth Caous, Maja S. Hellsing, Lovisa Ringstad, and Martin Anderssson ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01826 • Publication Date (Web): 23 May 2019 Downloaded from http://pubs.acs.org on May 25, 2019
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Applied Materials & Interfaces
Peptide-Loaded Cubosomes Functioning as an Antimicrobial Unit Against Escherichia coli Lukas Boge*1,5, Kathryn L. Browning2, Randi Nordström3, Mario Campana4, Liv S.E. Damgaard2, Josefin Seth Caous1, Maja Hellsing1, Lovisa Ringstad1, Martin Andersson5 1RISE
Research Institutes of Sweden, Borås, Sweden, 2Department of Pharmacy, University of Copenhagen, Denmark, 3 Department of Pharmacy, Uppsala University, Sweden, 4 Rutherford Appleton Laboratory, Didcot, United Kingdom, 5 Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Göteborg, Sweden. *Corresponding author,
[email protected] Abstract Dispersions of cubic liquid crystalline phases, also known as cubosomes, have shown great promise as delivery vehicles for a wide range of medicines. Due to their ordered structure, comprising alternating hydrophilic and hydrophobic domains, cubosomes possesses unique delivery properties and compatibility with both water soluble and insoluble drugs. However, the drug delivery mechanism and cubosome interaction with human cells and bacteria are still poorly understood. Herein, we reveal how cubosomes loaded with the human cathelicidin antimicrobial peptide LL-37, a system with high bacteria killing effect, interact with the bacterial membrane and provide new insights into the eradication mechanism. Combining the advanced experimental techniques neutron reflectivity and quartz crystal microbalance with dissipation monitoring a mechanistic drug delivery model for LL-37 loaded cubosomes on bacterial mimicking bilayers was constructed. Moreover, the cubosome interaction with Escherichia coli was directly visualized using super resolution laser scanning microscopy and cryogenic electron tomography. We could conclude that cubosomes loaded with LL-37 adsorbed and distorted bacterial membranes, providing evidence that the peptide-loaded cubosomes function as an antimicrobial unit. Key words: cubosome, antimicrobial peptide, LL-37, bacteria, membrane
Introduction Bacterial resistance to commercially available antibiotics is one of the greatest challenges for global health.1 Decades of misuse and overuse of antibiotics has accelerated resistance development leading to the demand for novel and efficient antibiotics to increase exponentially. Host defense peptides, such as antimicrobial peptides (AMPs), are present in virtually every form of life as part of the innate immune system and could be one alternative for the future treatment of bacterial infections.2-4 AMPs generally display a broad spectrum of antibacterial activity, with a low potential to induce resistance, shown by their continued activity over millions of years of co-evolution with bacteria.5 The AMP LL-37 is the only peptide in the cathelicidin family and is found in different cells, tissues and body fluids in humans.6 It displays beneficial broad-spectrum antibacterial properties; however, it is susceptible to proteolytic degradation by various human and bacterial elastases limiting therapeutic use. 7,8 The secondary structure of LL-37 normally changes from mainly random coil in aqueous solution to α-helix upon membrane adsorption, especially in the presence of negatively charged lipids and lipopolysaccharides (LPS).5,9,10
ACS Paragon Plus Environment
ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Self-assemblies of polar lipids, also known as lyotropic liquid crystals, have attracted great attention since their discovery decades ago.11-13 Recent studies have shown that LL-37 can be incorporated in dispersions of glycerol monooleate based liquid crystalline nanoparticles, known as cubosomes, displaying promising bactericidal activity in vitro.14-17 Previous investigations of the antibacterial properties of cubosomes loaded with LL-37 displayed a minimum inhibitory concentration (MIC) of 16 µg/mL against Escherichia coli (E. coli) with a slightly faster bacterial killing during the first 2-3 h in time-kill assays, compared to pure peptide at the same concentration.15,16 Pure cubosomes without peptide did not display any antibacterial properties. Moreover, cubosomes were also shown to successfully protect LL-37 from enzymatic degradation after exposure to infection related elastases.14,17 After enzyme exposure, the cubosomes loaded with LL-37 showed a significantly improved antibacterial effect, using radial diffusion assay, against E. coli, Staphylococcus aureus and Pseudomonas aeruginosa compared to pure peptide exposed to the same treatment.14,17 Cubosomes have been previously shown to not induce skin irritation, hence making them suitable for topical delivery.17 In order to form cubosomes, a liquid crystalline cubic phase is dispersed in the presence of a stabilizer.18-21 The interior of the cubosome particle is constituted of two bicontinuous, but nonintersecting, water channel systems separated by lipid bilayers.22,23 The bilayers are arranged in thermodynamically minimized three dimensional structures, representing infinite periodic minimal surfaces, with zero mean curvature.24 The well-organized internal structure and compatibility with hydrophilic, hydrophobic and amphiphilic molecules have made cubosomes widely explored as drug delivery vehicles.25-27 However, the bacteria killing mechanism of LL-37 loaded cubosomes is not well understood. As LL-37 is typically strongly associated with the cubosome, resulting in low and slow release of peptide,14,15,17 it can be hypothesized that the LL-37 and cubosome collectively function as an antimicrobial unit. In this study, we have investigated how cubosomes interact with model bacterial membranes, consisting of a mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoylsn-glycero-3-phospho-(1'-rac-glycerol) (DMPG), and E. coli bacteria. Quartz crystal microbalance with dissipation monitoring (QCM-D) and neutron reflectivity (NR) techniques were utilized to obtain detailed structural information of the cubosome-membrane interaction. Cubosome interactions with E. coli were visualized using super resolution laser scanning microscopy and cryogenic electron microscopy and tomography (cryo-TEM/ET). The combination of analytical tools allowed us to capture the cubosome-membrane interface ranging from Å to µm resolution thereby providing detailed information about the LL-37 loaded cubosome’s interaction with bacteria.
Experimental Materials Glycerol monoleate (GMO) Capmul-90 EP/NF was obtained from Abitec Corp. (Columbus, USA), the triblock co-polymeric stabilizer Poloxamer 407 (Kolliphor P407, approx. 12.500 g/mol, also known as Pluronic F-127) was obtained from BASF (Ludwigshafen, Germany), the water soluble antimicrobial peptide LL-37 (amino acid sequence: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES, molecular weight 4494 Da), purity 94.7%, was synthesized and provided by PolyPeptide Laboratories (Limhamn, Sweden), poly-L-lysine hydrobromide MW 30-70 kDa and D2O 99.99 atom % was from Sigma-Aldrich (St. Louis, USA). Lipids C14:0 1,2-dimyristoyl-sn-glycero-3-phosphocholine (hDMPC), 1,2-dimyristoylsn-glycero-3-phospho-(1'-rac-glycerol) sodium salt (hDMPG) and their tail-deuterated analogues 1,2dimyristoyl-d54-sn-glycero-3-phosphocholine (dDMPC) and 1,2-dimyristoyl-d54-sn-glycero-3-
ACS Paragon Plus Environment
Page 2 of 21
Page 3 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Applied Materials & Interfaces
[phospho-rac-(1-glycerol)] sodium salt (dDMPG) were purchased as powders from Avantii Polar Lipids Inc. (Alabaster, USA). Ultrapure water 18.2 Ω/cm (Milli-Q®) was used in all experiments for preparation of buffers and solutions. All chemicals were used as received. Methods Sample preparation Cubosomes with and without LL-37 were prepared by sonication of bulk cubic liquid crystalline gels.15 The ratio of GMO to water was 70:30, by weight. To enable fluorescent microscopy imaging, the florescent dye octadecyl rhodamine B R18 (Molecular Probes, Eugene, USA) was added to the cubosomes at a concentration of 0.2 mg/g GMO. Particle dispersions were prepared in 5 mM sodium acetate buffer pH 5.5 containing 0.5 wt % P407, at a cubosome concentration of 50 mg/mL with or without 0.5 mg/mL LL-37. The characteristics of the particles (size, size distribution, ζ-potential and structure) are summarized in Table S1 in Supporting information. Liposome preparation for bilayer formation Liposomes were formed by sonication of hydrated lipid films. Lipid films were prepared by dissolving the lipids (hDMPC and hDMPG or dDMPC and dDMPG) in chloroform at a molar ratio of 75:25 DMPC:DMPG in small glass vials. The lipid films were dried in a rotary evaporator at 40°C for 1 h, before being placed in a vacuum oven at room temperature overnight. Each film was hydrated to 1 mg/mL lipid at 40 °C in a water bath for 1 h and vortexed every 15 min followed by pulse sonication for 15-30 min using tip sonicators VibraCell VC 750 (Sonics and Materials Inc., USA) for hydrogenated lipids or a UP50H Ultrasonic Processor (Hielscher, Germany) for deuterated lipids. Quartz crystal microbalance with dissipation monitoring Quartz crystal microbalance with dissipation monitoring (E1, Q-Sense, Sweden) was used to study the interaction of cubosomes and LL-37 with supported bacterial mimicking bilayers. Silicon dioxide sensor chips (Q-sense, Sweden) were used in the experiments. After recording a stable baseline (
