Perspectives on nanomaterial-empowered bioremediation of heavy metals by photosynthetic microorganisms.

Environmental remediation of heavy metals (HMs) is a crucial aspect of sustainable development, safeguarding natural resources, biodiversity, and the delicate balance of ecosystems, all of which are critical for sustaining life on our planet. The bioremediation of HMs by unicellular phototrophs harnesses their intrinsic detoxification mechanisms, including biosorption, bioaccumulation, and biotransformation. These processes can be remarkably effective in mitigating HMs, particularly at lower contaminant concentrations, surpassing the efficacy of conventional physicochemical methods and offering greater sustainability and cost-effectiveness. Here, we explore the potential of various engineered nanomaterials to further enhance the capacity and efficiency of HM bioremediation based on photosynthetic microorganisms. The critical assessment of the interactions between nanomaterials and unicellular phototrophs emphasised the ability of tailored nanomaterials to sustain photosynthetic metabolism and the defence system of microorganisms, thereby enhancing their growth, biomass accumulation, and overall bioremediation capacity. Key factors that could shape future research efforts toward sustainable nanobioremediation of HM are discussed, and knowledge gaps in the field have been identified. This study sheds light on the potential of nanobioremediation by unicellular phototrophs as an efficient, scalable, and cost-effective solution for HM removal.

Living Diatom Microalgae for Desiccation-Resistant Electrodes in Biophotovoltaic Devices.

Strategies of renewable energy production from photosynthetic microorganisms are gaining great scientific interest as ecosustainable alternatives to fossil fuel depletion. Green microalgae have been thoroughly investigated as living components to convert solar energy into photocurrent in biophotovoltaic (BPV) cells. Conversely, the suitability of diatoms in BPV cells has been almost completely unexplored so far, despite being the most abundant class of photosynthetic microorganisms in phytoplankton and of their good adaptability and resistance to harsh environmental conditions, including dehydration, high salinity, nutrient starvation, temperature, or pH changes. Here, we demonstrate the suitability of a series of diatom species (Phaeodactylum tricornutum, Thalassiosira weissflogii, Fistulifera pelliculosa, and Cylindrotheca closterium), to act as biophotoconverters, coating the surface of indium tin oxide photoanodes in a model BPV cell. Effects of light intensity, cell density, total chlorophyll content, and concentration of the electrochemical mediator on photocurrent generation efficiency were investigated. Noteworthily, biophotoanodes coated with T. weissflogii diatoms are still photoactive after 15 days of dehydration and four rewetting cycles, contrary to analogue electrodes coated with the model green microalga Dunaliella tertiolecta. These results provide the first evidence that diatoms are suitable photosynthetic microorganisms for building highly desiccation-resistant biophotoanodes for durable BPV devices.

Sensitivity of Xylella fastidiosa subsp. pauca Salento-1 to light at 410 nm.

All over the world, from America to the Mediterranean Sea, the plant pathogen Xylella fastidiosa represents one of the most difficult challenges with many implications at ecological, agricultural, and economic levels. X. fastidiosa is a rod-shaped Gram-negative bacterium belonging to the family of Xanthomonadaceae. It grows at very low rates and infects a wide range of plants thanks to different vectors. Insects, through their stylets, suck a sap rich in nutrients and inject bacteria into xylem vessels. Since, until now, no antimicrobial treatment has been successfully applied to kill X. fastidiosa and/or prevent its diffusion, in this study, antimicrobial blue light (aBL) was explored as a potential anti-Xylella tool. Xylella fastidiosa subsp. pauca Salento-1, chosen as a model strain, showed a certain degree of sensitivity to light at 410 nm. The killing effect was light dose dependent and bacterial concentration dependent. These preliminary results support the potential of blue light in decontamination of agricultural equipment and/or plant surface; however, further investigations are needed for in vivo applications.

Polydopamine-Coated Liposomes for Methylene Blue Delivery in Anticancer Photodynamic Therapy: Effects in 2D and 3D Cellular Models.

Photodynamic therapy (PDT) is a therapeutic option for cancer, in which photosensitizer (PS) drugs, light, and molecular oxygen generate reactive oxygen species (ROS) and induce cell death. First- and second-generation PSs presented with problems that hindered their efficacy, including low solubility. Thus, second-generation PSs loaded into nanocarriers were produced to enhance their cellular uptake and therapeutic efficacy. Among other compounds investigated, the dye methylene blue (MB) showed potential as a PS, and its photodynamic activity in tumor cells was reported even in its nanocarrier-delivered form, including liposomes. Here, we prepared polydopamine (PDA)-coated liposomes and efficiently adsorbed MB onto their surface. lipoPDA@MB vesicles were first physico-chemically characterized and studies on their light stability and on the in vitro release of MB were performed. Photodynamic effects were then assessed on a panel of 2D- and 3D-cultured cancer cell lines, comparing the results with those obtained using free MB. lipoPDA@MB uptake, type of cell death induced, and ability to generate ROS were also investigated. Our results show that lipoPDA@MB possesses higher photodynamic potency compared to MB in both 2D and 3D cell models, probably thanks to its higher uptake, ROS production, and apoptotic cell death induction. Therefore, lipoPDA@MB appears as an efficient drug delivery system for MB-based PDT.

A simple strategy based on ATR-FTIR difference spectroscopy to monitor substrate intake and metabolite release by growing bacteria.

Attenuated total reflectance Fourier transform infrared (ATR-FTIR) difference spectroscopy has been employed for a variety of applications spanning from reaction mechanisms analysis to interface phenomena assessment. This technique is based on the detection of spectral changes induced by the chemical modification of the original sample. In the present study, we highlight the potential of the ATR-FTIR difference approach in the field of microbial biochemistry and biotechnology, reporting on the identification of main soluble species consumed and released by growing bacteria during the biohydrogen production process. Specifically, the mid-infrared spectrum of a model culture broth, composed of glucose, malt extract and yeast extract, was used as background to acquire the FTIR difference spectrum of the same broth as modified by Enterobacter aerogenes metabolism. The analysis of difference signals revealed that only glucose is degraded during hydrogen evolution in anaerobic conditions, while ethanol and 2,3-butanediol are the main soluble metabolites released with H2. This fast and easy analytical approach can therefore represent a sustainable strategy to screen different bacterial strains and to select raw and waste materials to be employed in the field of biofuel production.

Purification of Recombinant Human Amphiphysin 1 and its N-BAR Domain.

Bin/Amphiphysin/Rvs (BAR) proteins are known as classical membrane curvature generators during endocytosis. Amphiphysin, a member of the N-BAR sub-family of proteins that contain a characteristic amphipathic sequence at the N-terminus of the BAR domain, is involved in clathrin-mediated endocytosis. Full-length amphiphysin contains a ~ 400 amino acid long disordered linker connecting the N-BAR domain and a C-terminal Src homology 3 (SH3) domain. We express and purify recombinant amphiphysin and its N-BAR domain along with an N-terminal glutathione-S-transferase (GST) tag. The GST tag allows extraction of the protein of interest using affinity chromatography and is removed in the subsequent protease treatment and ion-exchange chromatography steps. In the case of the N-BAR domain, cleavage of the GST tag was found to cause precipitation. This issue can be minimized by adding glycerol to the protein purification buffers. In the final step, size exclusion chromatography removes any potential oligomeric species. This protocol has also been successfully used to purify other N-BAR proteins, such as endophilin, Bin1, and their corresponding BAR domains. Graphical overview.

Supramolecular Biohybrid Construct for Photoconversion Based on a Bacterial Reaction Center Covalently Bound to Cytochrome c by an Organic Light Harvesting Bridge.

A supramolecular construct for solar energy conversion is developed by covalently bridging the reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides and cytochrome c (Cyt c) proteins with a tailored organic light harvesting antenna (hCy2). The RC-hCy2-Cyt c biohybrid mimics the working mechanism of biological assemblies located in the bacterial cell membrane to convert sunlight into metabolic energy. hCy2 collects visible light and transfers energy to the RC, increasing the rate of photocycle between a RC and Cyt c that are linked in such a way that enhances proximity without preventing protein mobility. The biohybrid obtained with average 1 RC/10 hCy2/1.5 Cyt c molar ratio features an almost doubled photoactivity versus the pristine RC upon illumination at 660 nm, and ∼10 times higher photocurrent versus an equimolar mixture of the unbound proteins. Our results represent an interesting insight into photoenzyme chemical manipulation, opening the way to new eco-sustainable systems for biophotovoltaics.

Phytochemical Profiling and Untargeted Metabolite Fingerprinting of the MEDWHEALTH Wheat, Barley and Lentil Wholemeal Flours.

An important research target is improving the health benefits of traditional Mediterranean, durum wheat-based foods using innovative raw materials. In this study, we characterised wholemeal flours obtained from a traditional durum wheat cv. Svevo, two innovative durum wheat varieties (Svevo-High Amylose and Faridur), the naked barley cv. Chifaa and the elite lentil line 6002/ILWL118/1-1, evaluating them for targeted phytochemicals, untargeted metabolomics fingerprints and antioxidant capacity. To this aim, individual phenolic acids, flavonoids, tocochromanols and carotenoids were identified and quantified through HPLC-DAD, and the antioxidant capacities of both the extracts and whole meals were detected by ABTS assays. An untargeted metabolomics fingerprinting of the samples was conducted through NMR spectroscopy. Results showed that the innovative materials improved phytochemical profiles and antioxidant capacity compared to Svevo. In particular, Svevo-HA and Faridur had higher contents of ferulic and sinapic acids, ß-tocotrienol and lutein. Moreover, Chifaa is a rich source of phenolic acids, ß-tocopherols, lutein and zeaxanthin whereas lentil of flavonoids (i.e., catechin and procyanidin B2). The NMR profiles of Svevo-HA and Faridur showed a significant reduction of sugar content, malate and tryptophan compared to that of Svevo. Finally, substantial differences characterised the lentil profiles, especially for citrate, trigonelline and phenolic resonances of secondary metabolites, such as catechin-like compounds. Overall, these results support the potential of the above innovative materials to renew the health value of traditional Mediterranean durum wheat-based products.

Qualitative Characterization of Unrefined Durum Wheat Air-Classified Fractions.

Durum wheat milling is a key process step to improve the quality and safety of final products. The aim of this study was to characterize three bran-enriched milling fractions (i.e., F250, G230 and G250), obtained from three durum wheat grain samples, by using an innovative micronization and air-classification technology. Milling fractions were characterized for main standard quality parameters and for alveographic properties, starch composition and content, phenolic acids, antioxidant activity and ATIs. Results showed that yield recovery, ash content and particle size distributions were influenced either by the operating conditions (230 or 250) or by the grain samples. While total starch content was lower in the micronized sample and air-classified fractions, the P/L ratio increased in air-classified fractions as compared to semolina. Six main individual phenolic acids were identified through HPLC-DAD analysis (i.e., ferulic acid, vanillic acid, p-coumaric acid, sinapic acid, syringic and p-hydroxybenzoic acids). Compared to semolina, higher contents of all individual phenolic components were found in all bran-enriched fractions. The highest rise of TPAs occurred in the F250 fraction, which was maintained in the derived pasta. Moreover, bran-enriched fractions showed significant reductions of ATIs content versus semolina. Overall, our data suggest the potential health benefits of F250, G230 and G250 and support their use to make durum-based foods.

Chemical and morphological effects of the contraceptive hormone 17 α-ethynylestradiol on fluid lipid membranes.

The lack of studies involving the effects in human health associated with the chronic ingestion of pollutants lead to the path of investigating the action of these compounds in cell membrane models. We demonstrated the interaction (causes and consequences) of the hormone 17 α-ethinylestradiol (EE2) with lipid monolayers (prepared as Langmuir films) and bilayers prepared as small unilamellar vesicles (SUVs) and giant unilamellar vesicles (GUVs). Both fluidity and majority chemical composition of real plasma cell membrane were guaranteed using the phospholipid 1-palmitoil-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). Surface pressure-mean molecular area (π-A) isotherms and PM-IRRAS measurements highlighted the strong interaction of EE2 with POPC monolayers, leading the hormone to remain at the air/water interface and promoting its penetration into the phospholipid hydrophobic chains. In the case of bilayers, the entrance of the hormone inside the SUV is likely facilitated by their high curvature. In GUVs, EE2 was responsible for changes in the spherical shape, forming structures like buds and lipid protrusions. The set of results indicates the strong effects of EE2 on fluid membranes, which is an important feature to predict its damage in human cells.

Recent Advancements in Polymer/Liposome Assembly for Drug Delivery: From Surface Modifications to Hybrid Vesicles.

Liposomes are consolidated and attractive biomimetic nanocarriers widely used in the field of drug delivery. The structural versatility of liposomes has been exploited for the development of various carriers for the topical or systemic delivery of drugs and bioactive molecules, with the possibility of increasing their bioavailability and stability, and modulating and directing their release, while limiting the side effects at the same time. Nevertheless, first-generation vesicles suffer from some limitations including physical instability, short in vivo circulation lifetime, reduced payload, uncontrolled release properties, and low targeting abilities. Therefore, liposome preparation technology soon took advantage of the possibility of improving vesicle performance using both natural and synthetic polymers. Polymers can easily be synthesized in a controlled manner over a wide range of molecular weights and in a low dispersity range. Their properties are widely tunable and therefore allow the low chemical versatility typical of lipids to be overcome. Moreover, depending on their structure, polymers can be used to create a simple covering on the liposome surface or to intercalate in the phospholipid bilayer to give rise to real hybrid structures. This review illustrates the main strategies implemented in the field of polymer/liposome assembly for drug delivery, with a look at the most recent publications without neglecting basic concepts for a simple and complete understanding by the reader.

Supplementary Light Differently Influences Physico-Chemical Parameters and Antioxidant Compounds of Tomato Fruits Hybrids.

One of the challenges for agriculture in the coming years will be producing more food avoiding reducing the nutritional values of fruits and vegetables, sources of nutraceutical compounds. It has been demonstrated that light-emitting diodes (LEDs) used as a supplementary light (SL) technology improve tomato yield in Mediterranean greenhouses, but few data have been reported about SL effects on fruit physio-chemical parameters. In this study, three tomato hybrid (F1) cultivars were grown for year-round production in a commercial semi-closed glasshouse in Southern Italy: red cherry type ("Sorentyno"), red plum type ("Solarino"), and yellow plum type ("Maggino"). From 120 to 243 days after transplant (DAT), Red/White/Blue LEDs were used as SL. The fruits harvested 180 DAT were analyzed and those obtained under LEDs had 3% more dry weight, 15% more total soluble solids, and 16% higher titratable acidity than fruits grown only under natural light. Generally, the antioxidant activity and the mineral profile of the fruits were not negatively influenced by SL. Lycopene content was unchanged and vitamin C content of "Sorentyno" even increased by 15% under LEDs. Overall, LEDs used as SL technology could be one of the tools used by agriculture in Mediterranean basin to produce more food maintaining high quality production.

Exploiting the photoactivity of bacterial reaction center to investigate liposome dynamics.

Charge recombination kinetics of bacterial photosynthetic protein Reaction Center displays an exquisite sensitivity to the actual occupancy of ubiquinone-10 in its QB-binding site. Here, we have exploited such phenomenon for assessing the growth and the aggregation/fusion of phosphocholine vesicles embedding RC in their membrane, when treated with sodium oleate.

Chromatophores efficiently promote light-driven ATP synthesis and DNA transcription inside hybrid multicompartment artificial cells.

The construction of energetically autonomous artificial protocells is one of the most ambitious goals in bottom-up synthetic biology. Here, we show an efficient manner to build adenosine 5'-triphosphate (ATP) synthesizing hybrid multicompartment protocells. Bacterial chromatophores from Rhodobacter sphaeroides accomplish the photophosphorylation of adenosine 5'-diphosphate (ADP) to ATP, functioning as nanosized photosynthetic organellae when encapsulated inside artificial giant phospholipid vesicles (ATP production rate up to ∼100 ATP∙s-1 per ATP synthase). The chromatophore morphology and the orientation of the photophosphorylation proteins were characterized by cryo-electron microscopy (cryo-EM) and time-resolved spectroscopy. The freshly synthesized ATP has been employed for sustaining the transcription of a DNA gene, following the RNA biosynthesis inside individual vesicles by confocal microscopy. The hybrid multicompartment approach here proposed is very promising for the construction of full-fledged artificial protocells because it relies on easy-to-obtain and ready-to-use chromatophores, paving the way for artificial simplified-autotroph protocells (ASAPs).

Nanocellulose/Fullerene Hybrid Films Assembled at the Air/Water Interface as Promising Functional Materials for Photo-electrocatalysis.

Cellulose nanomaterials have been widely investigated in the last decade, unveiling attractive properties for emerging applications. The ability of sulfated cellulose nanocrystals (CNCs) to guide the supramolecular organization of amphiphilic fullerene derivatives at the air/water interface has been recently highlighted. Here, we further investigated the assembly of Langmuir hybrid films that are based on the electrostatic interaction between cationic fulleropyrrolidines deposited at the air/water interface and anionic CNCs dispersed in the subphase, assessing the influence of additional negatively charged species that are dissolved in the water phase. By means of isotherm acquisition and spectroscopic measurements, we demonstrated that a tetra-sulfonated porphyrin, which was introduced in the subphase as anionic competitor, strongly inhibited the binding of CNCs to the floating fullerene layer. Nevertheless, despite the strong inhibition by anionic molecules, the mutual interaction between fulleropyrrolidines at the interface and the CNCs led to the assembly of robust hybrid films, which could be efficiently transferred onto solid substrates. Interestingly, ITO-electrodes that were modified with five-layer hybrid films exhibited enhanced electrical capacitance and produced anodic photocurrents at 0.4 V vs Ag/AgCl, whose intensity (230 nA/cm2) proved to be four times higher than the one that was observed with the sole fullerene derivative (60 nA/cm2).

Tomato Oil Encapsulation by α-, ß-, and γ-Cyclodextrins: A Comparative Study on the Formation of Supramolecular Structures, Antioxidant Activity, and Carotenoid Stability.

Cyclodextrins (CDs) are oligosaccharides, comprising 6 (α), 7 (ß), or 8 (γ) glucose residues, used to prepare oil-in-water emulsions and improve oil stability towards degradation. In this research, the aptitude of α-, ß-, and γ-CDs to form complexes with a supercritical CO2 extracted lycopene-rich tomato oil (TO) was comparatively assessed. TO/CD emulsions and the resulting freeze-dried powders were characterized by microscopy, Fourier transform infrared-attenuated total reflection (FTIR-ATR), and differential scanning calorimetry (DSC), as well as for their antioxidant activity. Furthermore, carotenoid stability was monitored for 90 days at 25 and 4 °C. Confocal and SEM microscopy revealed morphological differences among samples. α- and ß-CDs spontaneously associated into microcrystals assembling in thin spherical shells (cyclodextrinosomes, Ø ≈ 27 µm) at the oil/water interface. Much smaller (Ø ≈ 9 µm) aggregates were occasionally observed with γ-CDs, but most TO droplets appeared "naked". FTIR and DSC spectra indicated that most CDs did not participate in TO complex formation, nevertheless structurally different interfacial complexes were formed. The trolox equivalent antioxidant capacity (TEAC) activity of emulsions and powders highlighted better performances of α- and ß-CDs as hydrophobic antioxidants-dispersing agents across aqueous media. Regardless of CDs type, low temperature slowed down carotenoid degradation in all samples, except all-[E]-lycopene, which does not appear efficiently protected by any CD type in the long storage period.

Oxygen Availability during Growth Modulates the Phytochemical Profile and the Chemo-Protective Properties of Spinach Juice.

Fruits and vegetables are a good source of potentially biologically active compounds. Their regular consumption in the human diet can help reduce the risk of developing chronic diseases such as cardiovascular diseases and cancer. Plants produce additional chemical substances when subject to abiotic stress or infected by microorganisms. The phytochemical profile of spinach leaves (Spinaciaoleracea L.), which is a vegetable with widely recognized health-promoting activity, has been affected by applying root hypoxic and re-oxygenation stress during plant growth. Leaf juice at different sampling times has been subject to liquid chromatography mass spectrometry (LC-MSn) analysis and tested on the human colorectal adenocarcinoma cell line HT29 by using the Comet assay. The cells were previously treated with H2O2 to simulate the presence of an oxidative stress (as in colon cancer condition) and the leaf juice application resulted in a significant antioxidant and protective in vitro effect. The duration of the hypoxic/re-oxygenation stress imposed on the plant reflects the antioxidant leaf juice content. After hypoxic stress (24 hours) and reoxygenation (2 hours), we show a decrease (50%) of the relative abundance of the principal identified antioxidant molecules but a higher antioxidant activity of the spinach juice on HT29 cells (20%). Data shows a complex relation between plant growing conditions and the modulation of secondary metabolites content in leaf juice that results in different chemo-protective activities in colon cancer cells.

A highly efficient heptamethine cyanine antenna for photosynthetic Reaction Center: From chemical design to ultrafast energy transfer investigation of the hybrid system.

The photosynthetic Reaction Center (RC) from the purple bacterium Rhodobacter sphaeroides has unique photoconversion capabilities, that can be exploited in assembly biohybrid devices for applications in solar energy conversion. Extending the absorption cross section of isolated RC through covalent functionalization with ad-hoc synthesized artificial antennas is a successful strategy to outperform the efficiency of the pristine photoenzyme under visible light excitation. Here we report a new heptamethine cyanine antenna that, upon covalent binding to RC, forms a biohybrid (hCyN7-RC) which, under white light excitation, has doubled photoconversion efficiency versus the bare photoenzyme. The artificial antenna hCyN7 successfully meets appropriate optical properties, i.e. peak position of absorption and emission maximum in the visible and NIR region respectively, large Stokes shift, and high fluorescence quantum yield, required for improving the efficiency of the biohybrid in the production of the charge-separated state in the RC. The kinetics of energy transfer and charge separation of hCyN7-RC studied via ultrafast visible and IR spectroscopies are here presented. The antenna transfers energy to RC chromophores within <10 ps and the rate of QA reduction is doubled compared to the native RC. These experiments further demonstrate hCyN7-RC, besides being an extremely efficient white light photoconverter, fully retains the charge separation mechanism and integrity of the native RC photoenzyme, thus allowing to envisage its suitability as biohybrid material in bioinspired systems for solar energy conversion.

Phosphate Modified Screen Printed Electrodes by LIFT Treatment for Glucose Detection.

The design of new materials as active layers is important for electrochemical sensor and biosensor development. Among the techniques for the modification and functionalization of electrodes, the laser induced forward transfer (LIFT) has emerged as a powerful physisorption method for the deposition of various materials (even labile materials like enzymes) that results in intimate and stable contact with target surface. In this work, Pt, Au, and glassy carbon screen printed electrodes (SPEs) treated by LIFT with phosphate buffer have been characterized by scanning electron microscopy and atomic force microscopy to reveal a flattening effect of all surfaces. The electrochemical characterization by cyclic voltammetry shows significant differences depending on the electrode material. The electroactivity of Au is reduced while that of glassy carbon and Pt is greatly enhanced. In particular, the electrochemical behavior of a phosphate LIFT treated Pt showed a marked enrichment of hydrogen adsorbed layer, suggesting an elevated electrocatalytic activity towards glucose oxidation. When Pt electrodes modified in this way were used as an effective glucose sensor, a 1⁻10 mM linear response and a 10 µM detection limit were obtained. A possible role of phosphate that was securely immobilized on a Pt surface, as evidenced by XPS analysis, enhancing the glucose electrooxidation is discussed.

Preparation of drug-loaded small unilamellar liposomes and evaluation of their potential for the treatment of chronic respiratory diseases.

The aim of the present investigation was to evaluate the influence of liposome formulation on the ability of vesicles to penetrate a pathological mucus model obtained from COPD affected patients in order to assess the potential of such vesicles for the treatment of chronic respiratory diseases by inhalation. Therefore, Small Unilamellar Liposomes (PLAIN-LIPOSOMEs), Pluronic® F127-surface modified liposomes (PF-LIPOSOMEs) and PEG 2000PE-surface modified liposomes (PEG-LIPOSOMEs) were prepared using the micelle-to-vesicle transition (MVT) method and beclomethasone dipropionate (BDP) as model drug. The obtained liposomes showed diameters in the range of 40-65 nm, PDI values between 0.25 and 0.30 and surface electric charge essentially close to zero. The encapsulation efficiency was found to be dependent on the BDP/lipid ratio used and, furthermore, BDP-loaded liposomes were stable in size both at 37 °C and at 4 °C. All liposomes were not cytotoxic on H441 cell line as assessed by the MTT assay. The liposome uptake was evaluated through a cytofluorimetric assay that showed a non-significant reduction in the internalization of PEG-LIPOSOMEs as compared with PLAIN-LIPOSOMEs. The penetration studies of mucus from COPD patients showed that the PEG-LIPOSOMEs were the most mucus-penetrating vesicles after 27 h. In addition, PEG- and PF-LIPOSOMEs did not cause any effect on bronchoalveolar lavage fluid proteins after aerosol administration in the mouse. The results highlight that PEG-LIPOSOMEs show the most interesting features in terms of penetration through the pathologic sputum, uptake by airway epithelial cells and safety profile.