RESUMEN
Microgels offer opportunities for improved delivery of antimicrobial peptides (AMP). To contribute to a foundation for rational design of such systems, we here study the effects of electrostatics on the generation of peptide-carrying microgels. For this, alginate microgels loaded with polymyxin B and cross-linked by Ca2+, were formed by electrostatic complexation using a hydrodynamic focusing three-dimensional (3D)-printed micromixer, varying pH and component concentrations. The structure of the resulting composite nanoparticles was investigated by small-angle X-ray scattering, dynamic light scattering, and z-potential measurements, whereas peptide encapsulation and release was monitored spectrophotometrically. Furthermore, membrane interactions of these systems were assessed by dye leakage assays in model lipid vesicles. Our results indicate that charge contrast between polymyxin B and alginate during microgel formation affects particle size and network dimensions. In particular, while microgels prepared at maximum polymyxin B-alginate charge contrast at pH 5 and 7.4 are characterized by sharp interfaces, those formed at pH 9 are characterized by a more diffuse core, likely caused by a weaker peptide-polymer affinity, and a shell dominated by alginate that shrinks at high CaCl2 concentrations. Quantitatively, however, these effects were relatively minor, as were differences in peptide encapsulation efficiency and electrolyte-induced peptide release. This demonstrates that rather wide charge contrasts allow efficient complexation and particle formation, with polymyxin B encapsulated within the particle interior at low ionic strength, but released at high electrolyte concentration. As a consequence of this, peptide-mediated membrane destabilization were suppressed by microgel incorporation at low ionic strength, but regained after microgel disruption. After particle disruption at high ionic strength, however, some polymyxin B was found to remain bound to alginate chains from the disrupted composite microgel particles, resulting in partial loss in membrane interactions, compared to the free peptide.
Asunto(s)
Alginatos/química , Antibacterianos/administración & dosificación , Liposomas/química , Microgeles/química , Polimixina B/administración & dosificación , Antibacterianos/química , Bacterias/química , Materiales Biomiméticos/química , Liberación de Fármacos , Tamaño de la Partícula , Polimixina B/química , Electricidad EstáticaRESUMEN
Due to rapid development of bacterial resistance against antibiotics, an emerging health crisis is underway, where 'simple' infections may no longer be treatable. Antimicrobial peptides (AMPs) constitute a class of substances attracting interest in this context. So far, research on AMPs has primarily focused on the identification of potent and selective peptides, as well as on the action mode of such peptides. More recently, there has been an increasing awareness that the delivery of AMPs is challenging due to their size, net positive charge, amphiphilicity, and proteolytic susceptibility. Hence, successful development of AMP therapeutics will likely require also careful design of efficient AMP delivery systems. In the present brief review, we discuss microgels, as well as related polyelectrolyte complexes and macroscopic hydrogels, as delivery systems for AMPs. In doing so, key factors for peptide loading and release are outlined and exemplified, together with consequences of this for functional performance relating to antimicrobial effects and cell toxicity.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias/efectos de los fármacos , Infecciones Bacterianas/tratamiento farmacológico , Portadores de Fármacos/química , Hidrogeles/química , Microgeles/química , Animales , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/toxicidad , HumanosRESUMEN
As a result of increasing bacterial resistance against antibiotics, we are facing an emerging health crisis, in which 'simple' infections may no longer be treatable. One class of molecules attracting interest in this context is antimicrobial peptides (AMPs), and considerable research efforts have been directed to identifying selective and potent AMPs. In addition, since in vivo delivery of AMPs is challenging, there is an emerging awareness that successful development of AMP therapeutics can be facilitated by careful design of AMPs delivery systems. In the present overview, we discuss polyelectrolyte complexation as a strategy to deliver AMPs. In doing so, key factors for AMP-polyelectrolyte complexation are illustrated for AMP-polyelectrolyte nanoparticle formation, as well as for AMP incorporation in polyelectrolyte microgels and multilayer structures, and consequences of these for functional performance exemplified.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Polielectrolitos/química , LiofilizaciónRESUMEN
In an effort to contribute to research in scalable production systems for polymeric delivery systems loaded with antimicrobial peptides (AMPs), we here investigate effects of hydrodynamic flow conditions on microfluidic particle generation. For this purpose, rapid prototyping using 3D printing was applied to prepare micromixers with three different geometric designs, which were used to prepare Ca2+-cross-linked alginate microgels loaded with the AMP polymyxin B in a continuous process. Based on fluid dynamic simulations, the hydrodynamic flow patterns in the micromixers were designed to be either (i) turbulent with chaotic disruption, (ii) laminar with convective mixing, or (iii) convective with microvortex formation. The physicochemical properties of the microgels prepared with these micromixers were characterized by photon correlation spectroscopy, laser-Doppler micro-electrophoresis, small-angle x-ray scattering, and ellipsometry. The particle size and compactness were found to depend on the micromixer geometry: From such studies, particle size and compactness were found to depend on micromixer geometry, the smallest and most compact particles were obtained by preparation involving microvortex flows, while larger and more diffuse microgels were formed upon laminar mixing. Polymyxin B was found to be localized in the particle interior and to cause particle growth with increasing peptide loading. Ca2+-induced cross-linking of alginate, in turn, results in particle contraction. The peptide encapsulation efficiency was found to be higher than 80% for all investigated micromixer designs; the highest encapsulation efficiency observed for the smallest particles generated by microvortex-mediated self-assembly. Ellipsometry results for surface-immobilized microgels, as well as results on peptide encapsulation, demonstrated electrolyte-induced peptide release. Taken together, these findings demonstrate that rapid prototyping of microfluidics using 3D-printed micromixers offers promises for continuous manufacturing of AMP-loaded microgels. Although the micromixer combining turbulent flow and microvortexes was demonstrated to be the most efficient, all three micromixer designs were found to mediate self-assembly of small microgels displaying efficient peptide encapsulation. This demonstrates the robustness of employing 3D-printed micromixers for microfluidic assembly of AMP-loaded microgels during continuous production.
Asunto(s)
Técnicas Analíticas Microfluídicas , Péptidos/química , Impresión Tridimensional , Calcio/química , Diseño de Equipo , Geles/síntesis química , Geles/química , Tamaño de la Partícula , Cloruro de Sodio/química , Propiedades de SuperficieRESUMEN
Successful use of microgels as delivery systems of antimicrobial peptides (AMPs) requires control of factors determining peptide loading and release to/from the microgels as well as of membrane interactions of both microgel particles and released peptides. Addressing these, we here investigate effects of microgel charge density and conformationally induced peptide amphiphilicity on AMP loading and release using detailed nuclear magnetic resonance (NMR) structural studies combined with ellipsometry, isothermal titration calorimetry, circular dichroism, and light scattering. In parallel, consequences of peptide loading and release for membrane interactions and antimicrobial effects were investigated. In doing so, poly(ethyl acrylate-co-methacrylic acid) microgels were found to incorporate the cationic AMPs EFK17a (EFKRIVQRIKDFLRNLV) and its partially d-amino acid-substituted variant EFK17da (E(dF)KR(dI)VQR(dI)KD(dF)LRNLV). Peptide incorporation was found to increase with increasing with microgel charge density and peptide amphiphilicity. After microgel incorporation, which appeared to occur preferentially in the microgel core, NMR showed EFK17a to form a helix with pronounced amphiphilicity, while EFK17da displayed a folded conformation, stabilized by a hydrophobic hub consisting of aromatic/aromatic and aliphatic/aromatic interactions, resulting in much lower amphiphilicity. Under wide ranges of peptide loading, the microgels displayed net negative z-potential. Such negatively charged microgels do not bind to, nor lyse, bacteria-mimicking membranes. Instead, membrane disruption in these systems is mediated largely by peptide release, which in turn is promoted at higher ionic strength and lower peptide amphiphilicity. Analogously, antimicrobial effects against Escherichia coli were found to be dictated by peptide release. Taken together, the findings show that peptide loading, packing, and release strongly affect the performance of microgels as AMP delivery systems, effects that can be tuned by (conformationally induced) peptide amphiphilicity and by microgel charge density.
Asunto(s)
Polímeros/química , Antiinfecciosos , Geles , Interacciones Hidrofóbicas e Hidrofílicas , Conformación MolecularRESUMEN
Evaluating the toxicity of self-assembled protein states is a key step towards developing effective strategies against amyloidogenic pathologies such as Alzheimer's and Parkinson's diseases. Such analysis is directly connected to quantitatively probing the stability of the cellular membrane upon interaction with different protein states. Using a combination of spectroscopic techniques, morphological observations, and spectral analysis of membrane fluctuations, we identify different destabilisation routes for giant unilamellar vesicles interacting with native-like states, prefibrillar species and amyloid-like fibrils of α-lactalbumin. These effects range from substantially lowering the bending rigidity of the membranes to irreversible structural changes and complete disruption of the lipid bilayers. Our findings clearly indicate how the wide heterogeneity in structures occurring during protein aggregation can result in different destabilisation pathways, acting on different length scales and not limited to enhanced membrane permeability.