Effects of charge contrast and composition on microgel formation and interactions with bacteria-mimicking liposomes
Graphical abstract
Introduction
Antimicrobial peptides (AMPs) form a broad family of amphiphilic peptides, generally of net positive charge, and frequently being relatively rich in hydrophobic amino acids [1,2]. Driven by increasing antibiotics resistance in bacteria, AMPs are currently attracting interest as potential therapeutics, also against multi-resistant and other challenging strains [3,4]. While a number of mechanisms contribute to such antimicrobial effect, lysis of bacterial membranes is key for the action of AMPs [1,5,6]. Furthermore, some AMPs display additional functions, including anti-inflammatory [2] and anti-cancer effects [7], which again involve interactions with membranes and their constituents.
Much work has been devoted to the identification of selective AMPs and their further optimization, including screening approaches [8], generation of peptides by infection-induced proteolysis [9], tagging by aromatic amino acids [10], as well as other approaches [11,12]. In contrast to extensive efforts on AMP identification and optimization, considerable less attention has been paid to drug delivery aspects of AMPs [13]. However, since AMPs are relatively large, amphiphilic, and net positively charged, efficient delivery of AMPs represents a hurdle in the development of these towards therapeutics, which may potentially be overcome by drug delivery systems.
Due to a typically low degree of cross-linking, microgels frequently display responsiveness to various stimuli [[14], [15], [16]]. Within the context of the present study, microgel interactions of peptides/proteins have been found to depend on peptide properties such as length, charge, hydrophobicity, and secondary structure [16], but also on microgel charge density [17,18] and cross-linking density [19,20], as well as on ambient conditions such as temperature [21,22], pH [23,24], and ionic strength [17,25]. Compared to the emerging understanding of model peptide/microgel systems, microgel-based delivery systems for AMPs have received less attention [13]. However, a growing number of studies in this area have demonstrated advantageous effects of combining AMPs with microgels, including reduced toxicity [26,27], increased antimicrobial effect, stability against enzyme degradation [28], and promoted cell internalization for combating intracellularly located pathogens [4]. As such, microgels offer opportunities as AMP delivery systems in various contexts, including parenteral, topical, and pulmonary routes. The biological environment encountered in these will likely influence peptide loading and release, as well as functional consequences thereof, depending on the chemistry used in the microgel design. In a development setting, microgels therefore have to be combined with AMPs in a way that optimally matches the indication and administration route at hand.
While functional advantages of microgels as delivery systems for AMPs are becoming increasingly demonstrated, challenges remain in the production of such systems. Thus, suspension polymerization frequently results in polydisperse microgels, and peptide loading often needs to be done after microgel synthesis, precluding continuous production. Mold-based microfluidics, on the other hand, provide excellent control of microgel size and structure, but production cost and complexity for these devices remain challenges in scale-up of production [29,30]. Considering this, we previously investigated the generation of AMP-loaded microgels through electrostatic complexation between polymyxin B and alginate, reinforced by Ca2+ ‘cross-linking’, using a simple microfluidic approach based on three dimensional (3D)-printed micromixers. It was found that small particles (100–200 nm) and efficient peptide encapsulation (>80%) could be achieved using several different micromixer designs, but that the best performance was obtained by convective mixing in combination with microvortex formation [31].
In the present work, we extend on this prior study by investigating effects of composition and electrostatics on the properties of peptide-loaded microgels formed by alginate, polymyxin B, and Ca2+. Polymyxin B was chosen as a model AMP based on: (i) structural similarities between polymyxin B and some classes of naturally occurring AMPs (including circulins and octapepsins [32]), and (ii) linear amphiphilicity similarities with AMPs conjugated with either acyl chains or aromatic amino acid stretches [33]. In addition, the pronounced linear amphiphilicity of polymyxin B provides an attractive peptide-peptide interaction likely to facilitate particle formation and peptide incorporation. In investigating the resulting microgels, structural features of microgels generated using a hydrodynamic focusing 3D-printed micromixer were investigated by small-angle x-ray scattering (SAXS), dynamic light scattering, zeta potential measurements, while information on peptide encapsulation was obtained by light spectroscopy. Furthermore, membrane interactions of these systems were assessed by dye leakage assays in model lipid vesicles.
Section snippets
Materials
Sodium alginate (208,000 g mol−1), polymyxin B sulfate salt (Mw = 1385.61 g mol−1), and CaCl2 ∙ 2H2O were obtained from Sigma Aldrich (Stockholm, Sweden), whereas dioleoylphosphoethanolamine (DOPE; >99%) and dioleoylphosphoglycerol (DOPG; >99%) were from Avanti Polar Lipids (Alabster, USA). 5-Carboxyfluorescein (CF) and bicinchoninic acid (BCA) were both from Sigma-Aldrich and were of analytical grade, as were all other chemicals used. Ultrapure Milli-Q water (18.2 MΩ ∙ cm) was used throughout.
Calculation of charge contrast
Key resources table
Resource Identifier 5-Carboxyfluorescein CF Bicinchoninic acid BCA Dioleoylphosphoethanolamine DOPE Dioleoylphosphoglycerol DOPG
Charge contrast between alginate and polymyxin B
As shown in Fig. 3, the charge density of both alginate and polymyxin B varies with pH. Thus, alginate is characterized by a pKa of ~3.5 [47], and becomes increasingly negatively charged with pH above 3 due to ionization of its carboxylic acid groups. The charge of polymyxin B, in contrast, is dominated by its diamino butyric groups with an effective pKa of ~10 [48], and becomes increasingly positively charged below this pH. As a consequence, the charge contrast (charge density of alginate
Discussion
The present work aimed at providing information on the properties and functionality of AMP-loaded microgels, formed by electrostatic complexation, using a microfluidic approach previously investigated [31]. Although studies on the effects of AMP uptake and release to/from microgels, as well as consequences of this on microgel structure, have been reported for some systems in literature, the relation between such effects and membrane interactions has been studied only in a couple of previous
Conclusions
As demonstrated for polymyxin B in combination with alginate and Ca2+ as high affinity “cross-linker” of alginate, peptide-loaded microgels can be formed using a wide range of compositions, and in a continuous way, using a microfluidic approach based on 3D-printed micromixers for hydrodynamic focusing mixing with vortex formation. Small microgel particles (> ≈ 125 nm radius) can be readily obtained over wide ranges in pH, as well as concentrations of polymer, peptide, and CaCl2. While charge
Acknowledgements
This research was funded by the European Commission (Erasmus Mobility Grant for Students) (MST), the Novo Nordisk Foundation (Interdisciplinary Synergy programme grant number NNF15OC0016670) (SB), and the Leo Foundation (Leo Foundation Center for Cutaneous Drug Delivery; grant number 2016-11-01) (MM).
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2020, BiomaterialsCitation Excerpt :As such, in a bid to tackle these issues, Borro et al. recently reported a new method of synthesizing AMP-loaded microgels, where electrostatic complexes, reinforced by Ca2+ ‘cross-linkages’, were formed between polymyxin B and alginate through a simple microfluidic approach [323]. Extending on this work, they further investigated the effects of composition and electrostatics on the complexes [324]. In this study, polymyxin B-loaded microgel particles were synthesized at various pH (5.0, 7.4, 9.0).
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