RÉSUMÉ
In the present study, we investigate the combined interaction of mesoporous silica (SiO2) and photocatalytic titanium dioxide (TiO2) nanoparticles with lipid membranes, using neutron reflectometry (NR), cryo-transmission electron microscopy (cryo-TEM), fluorescence oxidation assays, dynamic light scattering (DLS), and ζ-potential measurements. Based on DLS, TiO2 nanoparticles were found to display strongly improved colloidal stability at physiological pH of skin (pH 5.4) after incorporation into either smooth or spiky ("virus-like") mesoporous silica nanoparticles at low pH, the latter demonstrated by cryo-TEM. At the same time, such matrix-bound TiO2 nanoparticles retain their ability to destabilize anionic bacteria-mimicking lipid membranes under UV-illumination. Quenching experiments indicated both hydroxyl and superoxide radicals to contribute to this, while NR showed that free TiO2 nanoparticles and TiO2 loaded into mesoporous silica nanoparticles induced comparable effects on supported lipid membranes, including membrane thinning, lipid removal, and formation of a partially disordered outer membrane leaflet. By comparing effects for smooth and virus-like mesoporous nanoparticles as matrices for TiO2 nanoparticles, the interplay between photocatalytic and direct membrane binding effects were elucidated. Taken together, the study outlines how photocatalytic nanoparticles can be readily incorporated into mesoporous silica nanoparticles for increased colloidal stability and yet retain most of their capacity for photocatalytic destabilization of lipid membranes, and with maintained mechanisms for oxidative membrane destabilization. As such, the study provides new mechanistic information to the widely employed, but poorly understood, practice of loading photocatalytic nanomaterials onto/into matrix materials for increased performance.
Sujet(s)
Nanoparticules , Silice , Catalyse , Lipides , TitaneRÉSUMÉ
Conjugation with poly(ethylene glycol) ("PEGylation") is a widely used approach for improving the therapeutic propensities of peptide and protein drugs through prolonging bloodstream circulation, reducing toxicity and immunogenicity, and improving proteolytic stability. In the present study, we investigate how PEGylation affects the interaction of host defense peptides (HDPs) with bacterial lipopolysaccharide (LPS) as well as HDP suppression of LPS-induced cell activation. In particular, we investigate the effects of PEGylation site for KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), a peptide displaying potent anti-inflammatory effects, primarily provided by its N-terminal part. PEGylation was performed either in the N-terminus, the C-terminus, or in both termini, keeping the total number of ethylene groups (n = 48) constant. Ellipsometry showed KYE28 to exhibit pronounced affinity to both LPS and its hydrophobic lipid A moiety. The PEGylated peptide variants displayed lower, but comparable, affinity for both LPS and lipid A, irrespective of the PEGylation site. Furthermore, both KYE28 and its PEGylated variants triggered LPS aggregate disruption. To investigate the peptide structure in such LPS complexes, a battery of nuclear magnetic resonance (NMR) methods was employed. From this, it was found that KYE28 formed a well-folded structure after LPS binding, stabilized by hydrophobic domains involving aromatic amino acids as well as by electrostatic interactions. In contrast, the PEGylated peptide variants displayed a less well-defined secondary structure, suggesting weaker LPS interactions in line with the ellipsometry findings. Nevertheless, the N-terminal part of KYE28 retained helix formation after PEGylation, irrespective of the conjugation site. For THP1-Xblue-CD14 reporter cells, KYE28 displayed potent suppression of LPS activation at simultaneously low cell toxicity. Interestingly, the PEGylated KYE28 variants displayed similar or improved suppression of LPS-induced cell activation, implying the underlying key role of the largely retained helical structure close to the N-terminus, irrespective of PEGylation site. Taken together, the results show that PEGylation of HDPs can be done insensitively to the conjugation site without losing anti-inflammatory effects, even for peptides inducing such effects through one of its termini.
Sujet(s)
Lipide A/composition chimique , Lipopolysaccharides/composition chimique , Peptides/composition chimique , Polyéthylène glycols , Lignée cellulaire , Régulation de l'expression des gènes/effets des médicaments et des substances chimiques , Hémolyse , Humains , Modèles moléculaires , Facteur de transcription NF-kappa B/génétique , Facteur de transcription NF-kappa B/métabolisme , Peptides/pharmacologie , Liaison aux protéines , Conformation des protéines , Facteur de transcription AP-1/génétique , Facteur de transcription AP-1/métabolismeRÉSUMÉ
Local as well as systemic therapy is often used to treat bacterial lung infections. Delivery of antibiotics to the vascular side of infected lung tissue using lung-targeting microspheres (MS) is a good alternative to conventional administration routes, allowing for localized high levels of antibiotics. This delivery route can also complement inhaled antibiotic therapy, especially in the case of compromised lung function. We prepared and characterized monodisperse poly(lactic-co-glycolic acid) (PLGA) MS loaded with levofloxacin using a flow-focusing glass microfluidic chip. In vitro characterization showed that the encapsulated LVX displayed a biphasic controlled release during 5 days and preserved its antibacterial activity. The MS degradation was investigated in vitro by cross-sectioning the MS using a focused ion beam scanning electron microscope and in vivo by histological examination of lung tissue from mice intravenously administered with the MS. The MS showed changes in the surface morphology and internal matrix, whereas the degradation in vivo was 3 times faster than that in vitro. No effect on the viability of endothelial and lung epithelial cells or hemolytic activity was observed. To evaluate the pharmacokinetics and biodistribution of the MS, complete quantitative imaging of the 111indium-labeled PLGA MS was performed in vivo with single-photon emission computed tomography imaging over 10 days. The PLGA MS distributed homogeneously in the lung capillaries. Overall, intravenous administration of 12 µm PLGA MS is suitable for passive lung targeting and pulmonary therapy.
Sujet(s)
Acide lactique , Acide polyglycolique , Administration par voie intraveineuse , Animaux , Poumon , Souris , Microsphères , Copolymère d'acide poly(lactique-co-glycolique) , Distribution tissulaireRÉSUMÉ
Medicines based on small interfering RNA (siRNA) are promising for the treatment of a number of lung diseases. However, efficient delivery systems and design of stable dosage forms are required for inhalation therapy, as well as cost-effective methods for manufacturing of the final product. In this study, a 3D-printed micromixer was used for preparation of siRNA-dendrimer nanocomplexes, which were subsequently processed into microparticle-based dry powders for inhalation using spray drying. By applying the disposable micromixer, nanocomplexes were prepared of an average hydrodynamic diameter comparable to that of nanocomplexes prepared by manual mixing, but with narrower size distribution and low batch-to-batch variation. The nanocomplexes were processed into nanoembedded microparticles using different saccharide excipients. Data showed that siRNA integrity and bioactivity are retained after processing, and nanocomplexes could be reconstituted from the dry powders. The amorphous saccharide excipients trehalose and inulin provided better stabilization than crystalline mannitol, and they enabled full reconstitution of the nanocomplexes. In particular, a binary mixture of trehalose and inulin showed optimal stabilization, and enhanced cellular uptake and gene silencing efficiency. This study demonstrates that inexpensive and scalable micromixers can be used to optimize the production of siRNA-dendrimer nanocomplexes, and they can be applied in combination with spray drying for the engineering of dry powder formulations suitable for delivery of siRNA to the therapeutic target site.