Inhalable porous particles as dual micro-nano carriers demonstrating efficient lung drug delivery for treatment of tuberculosis.

Inhalation therapy treating severe infectious disease is among the more complex and emerging topics in controlled drug release. Micron-sized carriers are needed to deposit drugs into the lower airways, while nano-sized carriers are of preference for cell targeting. Here, we present a novel and versatile strategy using micron-sized spherical particles with an excellent aerodynamic profile that dissolve in the lung fluid to ultimately generate nanoparticles enabling to enhance both extra- and intra-cellular drug delivery (i.e., dual micro-nano inhalation strategy). The spherical particles are synthesised through the condensation of nano-sized amorphous silicon dioxide resulting in high surface area, disordered mesoporous silica particles (MSPs) with monodispersed size of 2.43 µm. Clofazimine (CLZ), a drug shown to be effective against multidrug-resistant tuberculosis, was encapsulated in the MSPs obtaining a dry powder formulation with high respirable fraction (F.P.F. <5 µm of 50%) without the need of additional excipients. DSC, XRPD, and Nitrogen adsorption-desorption indicate that the drug was fully amorphous when confined in the nano-sized pores (9-10 nm) of the MSPs (shelf-life of 20 months at 4 °C). Once deposited in the lung, the CLZ-MSPs exhibited a dual action. Firstly, the nanoconfinement within the MSPs enabled a drastic dissolution enhancement of CLZ in simulated lung fluid (i.e., 16-fold higher than the free drug), increasing mycobacterial killing than CLZ alone (p = 0.0262) and reaching concentrations above the minimum bactericidal concentration (MBC) against biofilms of M. tuberculosis (i.e., targeting extracellular bacteria). The released CLZ permeated but was highly retained in a Calu-3 respiratory epithelium model, suggesting a high local drug concentration within the lung tissue minimizing risk for systemic side effects. Secondly, the micron-sized drug carriers spontaneously dissolve in simulated lung fluid into nano-sized drug carriers (shown by Nano-FTIR), delivering high CLZ cargo inside macrophages and drastically decreasing the mycobacterial burden inside macrophages (i.e., targeting intracellular bacteria). Safety studies showed neither measurable toxicity on macrophages nor Calu-3 cells, nor impaired epithelial integrity. The dissolved MSPs also did not show haemolytic effect on human erythrocytes. In a nutshell, this study presents a low-cost, stable and non-invasive dried powder formulation based on a dual micro-nano carrier to efficiently deliver drug to the lungs overcoming technological and practical challenges for global healthcare.

Nano-FTIR spectroscopy of surface confluent polydopamine films - What is the role of deposition time and substrate material?

Polydopamine (PDA) is a widely used anchoring layer for multiple purposes. While simple to prepare, PDA is characterized by high chemical and topological diversity, which can limit its versatility. Unraveling the formation mechanism and physicochemical properties of continuous confluent layer and adherent nanoparticles on the nanoscale is crucial to further extend the prospective applications of PDA. Utilizing nano-FTIR spectroscopy, we investigate layers of PDA on three different substrates (silicon/silicon dioxide, nitrogen-doped titanium oxide, and gold substrates) at varying times of deposition (ToD). We observed a good correlation between the nano-FTIR and macroscopic FTIR spectra that reflected the changes in the relative abundance of PDA and polymerization intermediates as ToD increased. To gain analytical power, we utilized the principal component analysis (PCA) and extracted additional information from the resulting loadings spectral curves and data distribution in the score plots. We revealed a higher variability of the spectra of ultrathin surface confluent layers compared to the adherent nanoparticles. While the spectra of nanoparticles showed no apparent dependency on either ToD or the substrate material, the spectra of layers were highly affected by the increasing ToD and exhibited a rise in the absorption of PDA. Concomitantly, the spectra of layers grouped according to the substrate material at the lowest ToD point to the fact that the substrate material affects the PDA's initial physicochemical structure. The observed separation gradually diminished with the increasing ToD as the PDA physicochemical structure became less influenced by the substrate material.

Isolation of Mixed Compositions of Cellulose Nanocrystals, Microcrystalline Cellulose, and Lignin Nanoparticles from Wood Pulps.

From a circular economy perspective, one-pot strategies for the isolation of cellulose nanomaterials at a high yield and with multifunctional properties are attractive. Here, the effects of lignin content (bleached vs unbleached softwood kraft pulp) and sulfuric acid concentration on the properties of crystalline lignocellulose isolates and their films are explored. Hydrolysis at 58 wt % sulfuric acid resulted in both cellulose nanocrystals (CNCs) and microcrystalline cellulose at a relatively high yield (>55%), whereas hydrolysis at 64 wt % gave CNCs at a lower yield (<20%). CNCs from 58 wt % hydrolysis were more polydisperse and had a higher average aspect ratio (1.5-2×), a lower surface charge (2×), and a higher shear viscosity (100-1000×). Hydrolysis of unbleached pulp additionally yielded spherical nanoparticles (NPs) that were <50 nm in diameter and identified as lignin by nanoscale Fourier transform infrared spectroscopy and IR imaging. Chiral nematic self-organization was observed in films from CNCs isolated at 64 wt % but not from the more heterogeneous CNC qualities produced at 58 wt %. All films degraded to some extent under simulated sunlight trials, but these effects were less pronounced in lignin-NP-containing films, suggesting a protective feature, but the hemicellulose content and CNC crystallinity may be implicated as well. Finally, heterogeneous CNC compositions obtained at a high yield and with improved resource efficiency are suggested for specific nanocellulose uses, for instance, as thickeners or reinforcing fillers, representing a step toward the development of application-tailored CNC grades.

Elucidating the fine-scale structural morphology of nanocellulose by nano infrared spectroscopy.

Nanoscale infrared (IR) spectroscopy and microscopy, enabling the acquisition of IR spectra and images with a lateral resolution of 20 nm, is employed to chemically characterize individual cellulose nanocrystals (CNCs) and cellulose nanofibrils (CNFs) to elucidate if the CNCs and CNFs consist of alternating crystalline and amorphous domains along the CNF/CNC. The high lateral resolution enables studies of the nanoscale morphology at different domains of the CNFs/CNCs: flat segments, kinks, twisted areas, and end points. The types of nanocellulose investigated are CNFs from tunicate, CNCs from cotton, and anionic and cationic wood-derived CNFs. All nano-FTIR spectra acquired from the different samples and different domains of the individual nanocellulose particles resemble a spectrum of crystalline cellulose, suggesting that the non-crystalline cellulose signal observed in macroscopic measurements of nanocellulose most likely originate from cellulose chains present at the surface of the nanocellulose particles.

Solid-state polymer adsorption for surface modification: The role of molecular weight.

HYPOTHESIS: Solid-state polymer adsorption offers a distinct approach for surface modification. These ultrathin, so-called Guiselin layers can easily be obtained by placing a polymer melt in contact with an interface, followed by a removal of the non-adsorbed layer with a good solvent. While the mechanism of formation has been well established for Guiselin layers, their stability, crucial from the perspective of materials applications, is not. The stability is a trade-off in the entropic penalty between cooperative detachment of the number of segments directly adsorbed on the substrate and consecutively pinned monomers. EXPERIMENTS: Experimental model systems of Guiselin layers of polystyrene (PS) on silicon wafers with native oxide layer on top were employed. The stability of the adsorbed layers was studied as a function of PS molecular weight and polydispersibility by various microscopic and spectroscopic tools as well as quasi-static contact angle measurements. FINDINGS: Adsorbed layers from low molecular weight PS were disrupted with typical spinodal decomposition patterns whereas high molecular weight (>500 kDa) PS resulted in stable, continuous layers. Moreover, we show that Guiselin layers offer an enticing way to modify a surface, as demonstrated by adsorbed PS that imparts a hydrophobic character to initially hydrophilic silicon wafers.

High-Resolution Microscopical Studies of Contact Killing Mechanisms on Copper-Based Surfaces.

The mechanisms of bacterial contact killing induced by Cu surfaces were explored through high-resolution studies based on combinations of the focused ion beam (FIB), scanning transmission electron microscopy (STEM), high-resolution TEM, and nanoscale Fourier transform infrared spectroscopy (nano-FTIR) microscopy of individual bacterial cells of Gram-positive Bacillus subtilis in direct contact with Cu metal and Cu5Zn5Al1Sn surfaces after high-touch corrosion conditions. This approach permitted subcellular information to be extracted from the bioinorganic interface between a single bacterium and Cu/Cu5Zn5Al1Sn surfaces after complete contact killing. Early stages of interaction between individual bacteria and the metal/alloy surfaces include cell leakage of extracellular polymeric substances (EPSs) from the bacterium and changes in the metal/alloy surface composition upon adherence of bacteria. Three key observations responsible for Cu-induced contact killing include cell membrane damage, formation of nanosized copper-containing particles within the bacteria cell, and intracellular copper redox reactions. Direct evidence of cell membrane damage was observed upon contact with both Cu metal and Cu5Zn5Al1Sn surfaces. Cell membrane damage permits copper to enter into the cell interior through two possible routes, as small fragmentized Cu2O particles from the corrosion product layer and/or as released copper ions. This results in the presence of intracellular copper oxide nanoparticles inside the cell. The nanosized particles consist primarily of CuO with smaller amounts of Cu2O. The existence of two oxidation states of copper suggests that intracellular redox reactions play an important role. The nanoparticle formation can be regarded as a detoxification process of copper, which immobilizes copper ions via transformation processes within the bacteria into poorly soluble or even insoluble nanosized Cu structures. Similarly, the formation of primarily Cu(II) oxide nanoparticles could be a possible way for the bacteria to deactivate the toxic effects induced by copper ions via conversion of Cu(I) to Cu(II).

The nature of self-assembled octadecylphosphonic acid (ODPA) layers on copper substrates.

HYPOTHESIS: The self-assembly of amphiphilic molecules onto solid substrates can result both in the formation of monolayers and multilayers. However, on oxidized and non-oxidized copper (Cu), only monolayer formation was reported for phosphonic acids possessing one phosphate head group. Here, the adsorption of octadecylphosphonic acid (ODPA) on Cu substrates through a self-assembly process was investigated with the initial hypothesis of monolayer formation. EXPERIMENTS: The relative amount of ODPA adsorbed on a Cu substrate was determined by infrared reflection/absorption spectroscopy (IRRAS) and by atomic force microscopy (AFM) investigations before and after ODPA deposition. X-ray photoelectron spectroscopy (XPS) with sputtering was used to characterize the nature of the layers. FINDINGS: The results show that the thickness of the ODPA layer increased with deposition time, and after 1 h a multilayer film with a thickness of some tens of nm was formed. The film was robust and required long-time sonication for removal. The origin of the film robustness was attributed to the release of Cu ions, resulting in the formation of Cu-ODPA complexes with Cu ions in the form of Cu(I). Preadsorbing a monolayer of octadecylthiol (ODT) onto the Cu resulted in no ODPA adsorption, since the release of Cu(I) ions was abolished.

Correction to Infrared Nanospectroscopy of Phospholipid and Surfactin Monolayer Domains.

[This corrects the article DOI: 10.1021/acsomega.7b01931.].

Propofol adsorption at the air/water interface: a combined vibrational sum frequency spectroscopy, nuclear magnetic resonance and neutron reflectometry study.

Propofol is an amphiphilic small molecule that strongly influences the function of cell membranes, yet data regarding interfacial properties of propofol remain scarce. Here we consider propofol adsorption at the air/water interface as elucidated by means of vibrational sum frequency spectroscopy (VSFS), neutron reflectometry (NR), and surface tensiometry. VSFS data show that propofol adsorbed at the air/water interface interacts with water strongly in terms of hydrogen bonding and weakly in the proximity of the hydrocarbon parts of the molecule. In the concentration range studied there is almost no change in the orientation adopted at the interface. Data from NR show that propofol forms a dense monolayer with a thickness of 8.4 Å and a limiting area per molecule of 40 Å2, close to the value extracted from surface tensiometry. The possibility that islands or multilayers of propofol form at the air/water interface is therefore excluded as long as the solubility limit is not exceeded. Additionally, measurements of the 1H NMR chemical shifts demonstrate that propofol does not form dimers or multimers in bulk water up to the solubility limit.

Infrared Nanospectroscopy of Phospholipid and Surfactin Monolayer Domains.

A main challenge in understanding the structure of a cell membrane and its interactions with drugs is the ability to chemically study the different molecular species on the nanoscale. We have achieved this for a model system consisting of mixed monolayers (MLs) of the biologically relevant phospholipid 1,2-distearoyl-sn-glycero-phosphatidylcholine and the antibiotic surfactin. By employing nano-infrared (IR) microscopy and spectroscopy in combination with atomic force microscopy imaging, it was possible to identify and chemically detect domain formation of the two constituents as well as to obtain IR spectra of these species with a spatial resolution on the nanoscale. A novel method to enhance the near-field imaging contrast of organic MLs by plasmon interferometry is proposed and demonstrated. In this technique, the organic layer is deposited on gold and ML graphene substrates, the latter of which supports propagating surface plasmons. Plasmon reflections arising from changes in the dielectric environment provided by the organic layer lead to an additional contrast mechanism. Using this approach, the interfacial region between surfactin and the phospholipid has been mapped and a transition region is identified.

Mixed monolayers of alkane thiols with polar terminal group on gold: Investigation of structure dependent surface properties.

Adsorption of thiols with cationic or anionic terminal group on gold has been studied from mixed solutions of 11-Amino-1-undecanethiol (AUT) and 3-Mercaptopropionic acid (MPA) using Quartz Crystal Microbalance with Dissipation (QCM-D), X-ray Photoelectron Spectroscopy (XPS), atomic force microscopy (AFM) and contact angles. The goal is to probe the nature of such layers, and the additivity or otherwise of the pH responsiveness, with a view to evaluate their suitability as smart materials. For each of the two pure (unmixed) cases, ordered molecular monolayers are formed with sulfur binding to gold and the alkane chain pointing out from the surface as expected. Adsorption from the thiol mixtures, however, leads to a more complex behaviour. The surface concentration of thiols from the mixtures, as determined by QCM-D, is considerably lower than for the pure cases and it reaches a minimum at a 3:1 MPA/AUT relative concentration in the solution. The XPS results confirm a reduction in adsorbed amount in mixtures with the lowest overall intensity for the 3:1 ratio. Monolayers formed from mixtures display a wettability which is much lower and less pH sensitive. Collectively these results confirm that for adsorption from mixed systems, the configuration is completely different. Complex formation in the mixed solutions leads to the adsorption of molecules parallel to the surface in an axially in-plane configuration. This parallel layer of thiols is mechanically relatively robust to nano-shaving based on AFM measurements. These results will have a significant impact on the design of biomimetic surface coatings particularly when mixtures of oppositely charged molecules are present on the surface, as is commonly the case in biological, proteinaceous surfaces (e.g. hair and skin).

Vibrational Sum Frequency Spectroscopy on Polyelectrolyte Multilayers: Effect of Molecular Surface Structure on Macroscopic Wetting Properties.

Adsorption of a single layer of molecules on a surface, or even a reorientation of already present molecules, can significantly affect the surface properties of a material. In this study, vibrational sum frequency spectroscopy (VSFS) has been used to study the change in molecular structure at the solid-air interface following thermal curing of polyelectrolyte multilayers of poly(allylamine hydrochloride) and poly(acrylic acid). Significant changes in the VSF spectra were observed after curing. These changes were accompanied by a distinct increase in the static water contact angle, showing how the properties of the layer-by-layer molecular structure are controlled not just by the polyelectrolyte in the outermost layer but ultimately by the orientation of the chemical constituents in the outermost layers.

Monolayer study by VSFS: in situ response to compression and shear in a contact.

Self-assembled octadecyltrichlorosilane ((OTS), CH3(CH2)17SiCl3) layers on hydroxyl-terminated silicon oxide (SiO2) were prepared. The monolayers were characterized with atomic force microscopy (AFM) and contact angle measurements; their conformation was studied before, during, and after contact with a polymer (either PDMS or PTFE) surface using the vibrational sum frequency spectroscopy (VSFS) technique. During contact, the effect of pressure was studied for both polymer surfaces, but in the case of PTFE, the effect of shear rate on the contact was simultaneously studied. The VSFS response of the monolayers with pressure was almost entirely due to changes in the real area of contact with the polymer and therefore the Fresnel factors, whereas sliding caused disorder in the previously all-trans monolayer, as evidenced by a significant increase in the population of gauche defects.

Combined in situ quartz crystal microbalance with dissipation monitoring, indirect nanoplasmonic sensing, and vibrational sum frequency spectroscopic monitoring of alkanethiol-protected copper corrosion.

In this study, we have applied three techniques to simultaneously and in situ study the initial stage of corrosion of copper protected by a self-assembled monolayer of octadecanethiol (ODT). We combined quartz crystal microbalance with dissipation monitoring (QCM-D), indirect nanoplasmonic sensing (INPS), and vibrational sum frequency spectroscopy (VSFS) and obtained complementary information about mass uptake and optical and spectroscopic changes taking place during the initial corrosion phase. All three techniques are very sensitive to the formation of a corrosion film (thickness in the range 0-0.41 nm) under mildly corrosive conditions (dry air, <0.5% relative humidity). The three techniques yield information about the viscoelasticity of the corrosion film (QCM-D), the homogeneity of the corrosion reaction on the surface (INPS), and the stability of the ODT protection layer (VSFS). Furthermore, by also studying the corrosion process in humid air (ca. 70% relative humidity), we illustrate how the combination of these techniques can be used to differentiate between simultaneously occurring processes, such as water adsorption and corrosion product formation.

Crown ethers at the aqueous solution-air interface. Part 2. Electrolyte effects, ethylene oxide hydration and temperature behaviour.

Vibrational Sum Frequency Spectroscopy (VSFS) was employed to study adsorbing films of 4-Nitro Benzo-15-Crown-5 (NB15C5) and Benzo-15-Crown-5 (B15C5) at the aqueous solution-air interface. The surface of the solution is strongly influenced by the presence of crown ether species. Changes in the orientation of NB15C5 were monitored as a function of solution concentration, by targeting the ratio of peak intensities of the CN and NO(2) vibrational modes. The water of hydration has also been probed as a function of crown concentration, salt concentration, and temperature. The latter study strongly suggests that the surface can be treated as a charged interface, and that the associated ordered water decreases with increasing ionic strength of the bulk solution.

Crown ethers at the aqueous solution-air interface: 1. Assignments and surface spectroscopy.

The surface of aqueous solutions of 4-Nitro Benzo-15-Crown-5 (NB15C5) and Benzo-15-Crown-5 (B15C5) has been studied using the surface sensitive technique vibrational sum frequency spectroscopy (VSFS). The NO, CN, COC and CH vibrational modes of these compounds at the air-water interface as well as OH vibrational modes of the surface water hydrating this compound have been targeted in order to obtain molecular information about arrangement and conformation of the adsorbed crown ether molecules at the air-water interface. The CH(2) vibrational modes of crown ethers have been identified and found to be split due to interaction with ether oxygen. The spectra provide evidence for the existence of a protonated crown complex moiety at the surface leading to the appearance of strongly ordered water species. The interfacial water species are influenced by the resulting charged interface and by the strong Zundel polarizability due to tunneling of the proton species between equivalent sites within the crown ring.

Phospholipid monolayers probed by vibrational sum frequency spectroscopy: instability of unsaturated phospholipids.

The surface specific technique vibrational sum frequency spectroscopy has been applied to in situ studies of the degradation of Langmuir monolayers of 1,2-diacyl-phosphocholines with various degrees of unsaturation in the aliphatic chains. To monitor the degradation of the phospholipids, the time-dependent change of the monolayer area at constant surface pressure and the sum frequency intensity of the vinyl CH stretch at the carbon-carbon double bonds were measured. The data show a rapid degradation of monolayers of phospholipids carrying unsaturated aliphatic chains compared to the stable lipids carrying fully saturated chains when exposed to the ambient laboratory air. In addition, the degradation of the phospholipids can be inhibited by purging the ambient air with nitrogen. This instability may be attributed to spontaneous degradation by oxidation mediated by various reactive species in the air. To further elucidate the process of lipid oxidation in biological membranes artificial Langmuir monolayers probed by a surface specific spectroscopic technique as in this study can serve as a model system for studying the degradation/oxidation of cell membrane constituents.

Optical sum-frequency emission from langmuir-blodgett films of variable thickness: effects of the substrate and polar orientation of fatty acids in the films.

The nonlinear optical response arising from a model multilayer structure, i.e., Langmuir-Blodgett (LB) films comprised of different numbers of per-protonated (H) and per-deuterated (D) fatty acid layers on solid substrates, has been evaluated by sum-frequency generation (SFG) spectroscopy. The SFG signals depend significantly on the absolute polar orientation of the fatty acids in the individual layers and on the nonlinear optical response of the substrate. The SFG spectra on gold and fused quartz substrates demonstrate a totally different dependence on the number of the contributing H layers, which it is possible to analyze quantitatively. The results provide important information for understanding the origin of the nonlinear optical responses from ordered systems composed of multiple interfaces and therefore for extracting exact structural information about each interface from the observed SFG signals.

Hydration state of nonionic surfactant monolayers at the liquid/vapor interface: structure determination by vibrational sum frequency spectroscopy.

The OH stretching region of water molecules in the vicinity of nonionic surfactant monolayers has been investigated using vibrational sum frequency spectroscopy (VSFS) under the polarization combinations ssp, ppp, and sps. The surface sensitivity of the VSFS technique has allowed targeting the few water molecules present at the surface with a net orientation and, in particular, the hydration shell around alcohol, sugar, and poly(ethylene oxide) headgroups. Dramatic differences in the hydration shell of the uncharged headgroups were observed, both in comparison to each another and in comparison to the pure water surface. The water molecules around the rigid glucoside and maltoside sugar rings were found to form strong hydrogen bonds, similar to those observed in tetrahedrally coordinated water in ice. In the case of the poly(ethylene oxide) surfactant monolayer a significant ordering of both strongly and weakly hydrogen bonded water was observed. Moreover, a band common to all the surfactants studied, clearly detected at relatively high frequencies in the polarization combinations ppp and sps, was assigned to water species located in proximity to the surfactant hydrocarbon tail phase, with both hydrogen atoms free from hydrogen bonds. An orientational analysis provided additional information on the water species responsible for this band.