Drug Delivery through Epidermal Tissue Cells by Functionalized Biosilica from Diatom Microalgae.

Diatom microalgae are a natural source of fossil biosilica shells, namely the diatomaceous earth (DE), abundantly available at low cost. High surface area, mesoporosity and biocompatibility, as well as the availability of a variety of approaches for surface chemical modification, make DE highly profitable as a nanostructured material for drug delivery applications. Despite this, the studies reported so far in the literature are generally limited to the development of biohybrid systems for drug delivery by oral or parenteral administration. Here we demonstrate the suitability of diatomaceous earth properly functionalized on the surface with n-octyl chains as an efficient system for local drug delivery to skin tissues. Naproxen was selected as a non-steroidal anti-inflammatory model drug for experiments performed both in vitro by immersion of the drug-loaded DE in an artificial sweat solution and, for the first time, by trans-epidermal drug permeation through a 3D-organotypic tissue that better mimics the in vivo permeation mechanism of drugs in human skin tissues. Octyl chains were demonstrated to both favour the DE adhesion onto porcine skin tissues and to control the gradual release and the trans-epidermal permeation of Naproxen within 24 h of the beginning of experiments. The evidence of the viability of human epithelial cells after permeation of the drug released from diatomaceous earth, also confirmed the biocompatibility with human skin of both Naproxen and mesoporous biosilica from diatom microalgae, disclosing promising applications of these drug-delivery systems for therapies of skin diseases.

Boronic Acid Moieties Stabilize Adhesion of Microalgal Biofilms on Glassy Substrates: A Chemical Tool for Environmental Applications.

Photosynthetic organisms such as diatoms microalgae provide innovative routes to eco-friendly technologies for environmental pollution bioremediation. Living diatoms are capable to incorporate in vivo a wide variety of chemical species dispersed in seawater, thus being promising candidates for eco-friendly removal of toxic contaminants. However, their exploitation requires immobilization methods that allow to confine microalgae during water treatment. Here we demonstrate that a biofilm of Phaeodactylum tricornutum diatom cells grown on the surface of a glassy substrate bearing boronic acid protruding moieties is stably anchored to the substrate resisting mechanical stress and it is suitable for removal of up to 80 % metal ions (As, Cr, Cu, Zn, Sn, Pb, Sb) in a model polluted water sample. Control experiments also suggest that stabilization of the biofilm adhesion occurs by interaction of boronic acid surface groups of the substrate with the hydroxyl groups of diatoms extracellular polysaccharides.

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.

Improving the In Vitro Removal of Indoxyl Sulfate and p-Cresyl Sulfate by Coating Diatomaceous Earth (DE) and Poly-vinyl-pyrrolidone-co-styrene (PVP-co-S) with Polydopamine.

Polydopamine (PDA) is a synthetic eumelanin polymer mimicking the biopolymer secreted by mussels to attach to surfaces with a high binding strength. It exhibits unique adhesive properties and has recently attracted considerable interest as a multifunctional thin film coating. In this study, we demonstrate that a PDA coating on silica- and polymer-based materials improves the entrapment and retention of uremic toxins produced in specific diseases. The low-cost natural nanotextured fossil diatomaceous earth (DE), an abundant source of mesoporous silica, and polyvinylpyrrolidone-co-Styrene (PVP-co-S), a commercial absorbent comprising polymeric particles, were easily coated with a PDA layer by oxidative polymerization of dopamine at mild basic aqueous conditions. An in-depth chemical-physical investigation of both the resulting PDA-coated materials was performed by SEM, AFM, UV-visible, Raman spectroscopy and spectroscopic ellipsometry. Finally, the obtained hybrid systems were successfully tested for the removal of two uremic toxins (indoxyl sulfate and p-cresyl sulfate) directly from patients' sera.

Polydopamine coating of living diatom microalgae.

Many microorganisms produce specific structures, known as spores or cysts, to increase their resistance to adverse environmental conditions. Scientists have started to produce biomimetic materials inspired by these natural membranes, especially for industrial and biomedical applications. Here, we present biological data on the biocompatibility of a polydopamine-based artificial coating for diatom cells. In this work, living Thalassiosira weissflogii diatom cells are coated on their surface with a polydopamine layer mimicking mussel adhesive protein. Polydopamine does not affect diatoms growth kinetics, it enhances their resistance to degradation by treatment with detergents and acids, and it decreases the uptake of model staining emitters. These outcomes pave the way for the use of living diatom cells bearing polymer coatings for sensors based on living cells, resistant to artificial microenvironments, or acting as living devices for cells interface study.

Incorporating a molecular antenna in diatom microalgae cells enhances photosynthesis.

Diatom microalgae have great industrial potential as next-generation sources of biomaterials and biofuels. Effective scale-up of their production can be pursued by enhancing the efficiency of their photosynthetic process in a way that increases the solar-to-biomass conversion yield. A proof-of-concept demonstration is given of the possibility of enhancing the light absorption of algae and of increasing their efficiency in photosynthesis by in vivo incorporation of an organic dye which acts as an antenna and enhances cells' growth and biomass production without resorting to genetic modification. A molecular dye (Cy5) is incorporated in Thalassiosira weissflogii diatom cells by simply adding it to the culture medium and thus filling the orange gap that limits their absorption of sunlight. Cy5 enhances diatoms' photosynthetic oxygen production and cell density by 49% and 40%, respectively. Cy5 incorporation also increases by 12% the algal lipid free fatty acid (FFA) production versus the pristine cell culture, thus representing a suitable way to enhance biofuel generation from algal species. Time-resolved spectroscopy reveals Förster Resonance Energy Transfer (FRET) from Cy5 to algal chlorophyll. The present approach lays the basis for non-genetic tailoring of diatoms' spectral response to light harvesting, opening up new ways for their industrial valorization.

Advanced Gel Permeation Chromatography system with increased loading capacity: Polycyclic aromatic hydrocarbons detection in olive oil as a case of study.

Gel permeation chromatography (GPC) is herein used as size exclusion clean-up technique for highly sensitive and straightforward detection of Polycyclic Aromatic Hydrocarbons (PAHs) in olive oil samples. An advanced chromatographic system has been developed to isolate a series of PAHs with cancerogenic potential, including PAH4 (benzo(a)pyrene BaP, benzo(a)anthracene BaA, benzo(b)fluoranthene BbF and chrysene Chry) reported in the European Regulation. The system avails of two glass chromatographic columns and a switching valve, that allow removal of interfering analytes in olive oil without resorting to any preliminary extraction process. A seven-fold increase of the loaded sample amount versus conventional chromatographic systems (1 g vs 0.150 g) has been pursued, as well as improved PAHs detection and quantification limits (LOD-LOQ for PAH4: 0.21-0.70 ng/g for BaA, 0.26-0.86 ng/g for Chry, 0.23-0.76 ng/g for BbF, 0.32-1.06 ng/g for BaP), in accordance with the continuous need of more and more reducing these limits in food analysis by the European Regulation. The protocol developed represents a highly innovative and efficient analytical method for organic pollutants in complex biological matrices as olive oil, that can have huge impact on technology for PAHs detection in food samples, being suitable for both industrial and small-scale laboratories.

Supercritical CO2 Extraction of Phytocompounds from Olive Pomace Subjected to Different Drying Methods.

Olive pomace is a semisolid by-product of olive oil production and represents a valuable source of functional phytocompounds. The valorization of agro-food chain by-products represents a key factor in reducing production costs, providing benefits related to their reuse. On this ground, we herein investigate extraction methods with supercritical carbon dioxide (SC-CO2) of functional phytocompounds from olive pomace samples subjected to two different drying methods, i.e., freeze drying and hot-air drying. Olive pomace was produced using the two most common industrial olive oil production processes, one based on the two-phase (2P) decanter and one based on the three-phase (3P) decanter. Our results show that freeze drying more efficiently preserves phytocompounds such as α-tocopherol, carotenoids, chlorophylls, and polyphenols, whereas hot-air drying does not compromise the ß-sitosterol content and the extraction of squalene is not dependent on the drying method used. Moreover, higher amounts of α-tocopherol and polyphenols were extracted from 2P olive pomace, while ß-sitosterol, chlorophylls, and carotenoids were more concentrated in 3P olive pomace. Finally, tocopherol and pigment/polyphenol fractions exerted antioxidant activity in vitro and in accelerated oxidative conditions. These results highlight the potential of olive pomace to be upcycled by extracting from it, with green methods, functional phytocompounds for reuse in food and pharmaceutical industries.

About the origin of the large Stokes shift in aminoalkyl substituted heptamethine cyanine dyes.

Aminoalkyl-substituted heptamethine cyanine dyes are characterized by a large Stokes shift, an uncommon feature for cyanine molecules yet very promising for their application as fluorescent probes in bioimaging and as light harvesting antennas in biohybrid systems for solar energy conversion. The origin of this photophysical feature has not been adequately explored so far, and a combined experimental and theoretical work is herein provided to shed light on the role played by the central aminoalkyl substituent bound to the heptamethine cyanine backbone in defining the unusual properties of the dye. The spectra recorded in solvents of different polarities point to a marginal role of the medium in the definition of the Stokes shift, which conversely can be ascribed to the relaxation of the molecular geometry upon photoexcitation. This hypothesis is supported by an extensive theoretical investigation of the ground and excited states of the dye. TD-DFT results on the aminoalkyl-substituted dye and its unsubstituted precursor demonstrate a very similar cyanine-like structure for both molecules in the relaxed excited state. Conversely, in the ground state the amino substitution disrupts the conjugation in the polymethine chain, leading to a broken-symmetry, non-planar structure.

In vivo functionalization of diatom biosilica with sodium alendronate as osteoactive material.

Bisphosphonates are a class of drugs widely used in the clinical treatment of disorders of bone metabolism, such as osteoporosis, fibrous dysplasia, myeloma and bone metastases. Because of the negative side effects caused by oral administration of bisphosphonates, various silica mesoporous materials have been investigated for a confined and controlled release of these drugs. Here, we propose biosilica from diatoms as suitable substrate for alendronate local activation of bone cells. Following a novel strategy, sodium alendronate can be in vivo incorporated into biosilica shells of cultured Thalassiosira weissflogii diatoms, by feeding the algae with an aqueous solution of the drug. After acid/oxidative treatments for removing organic matter, the resulting bisphosphonate-functionalized mesoporous biosilica was characterized and tested as osteoinductive support. Effects on osteoblast growth and anti-osteoclast activity have been examined by evaluating SaOS-2, BMSC, J774 cell viability on the alendronate-"doped" biosilica. The loading percentage of sodium alendronate into biosilica, estimated as 1.45% w/w via TGA, was able to decrease metabolic activity of J774 osteoclasts-like cells till 5% over glass control. We demonstrated a good osteoconductive ability and activation of a tissue regeneration model together with osteoclasts inhibition of the functionalized biosilica, opening the way to interesting applications for diatom microalgae as a bioinspired mesoporous material for tissue engineering.

Data from in vivo functionalization of diatom mesoporous biosilica with bisphosphonates.

Diatoms are unicellular photosynthetic microalgae that produce a sophisticated mesoporous biosilica shell called frustule. Easy to achieve and extract, diatom frustules represent a low-cost source of mesoporous biocompatible biosilica. In this paper, the possibility to in vivo functionalize the diatom biosilica with bisphosphonates (BPs) was investigated. In particular, two BPs were tested: the amino-containing sodium alendronate (ALE) and the amino-lacking sodium etidronate (ETI). According to first SEM-EDX analysis, the presence of the amino-moiety in ALE structure allowed a better incorporation of this BP into living diatom biosilica, compared to ETI. Then, diatom growth was deeply investigated in presence of ALE. After extraction of functionalized frustules, ALE-biosilica was further characterized by XPS and microscopy, and ALE release was evaluated by ferrochelation assay. Moreover, the bone regeneration performances of ALE-functionalized frustules were preliminarily investigated on bone osteoblast-like cells, via Comassie staining. Data are related to the research article "In vivo functionalization of diatom biosilica with sodium alendronate as osteoactive material".

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.

Solvent-Free Pd-Catalyzed Heteroaryl-Aryl Coupling via C-H Bond Activation for the Synthesis of Extended Heteroaromatic Conjugated Molecules.

Direct arylation of thienopyrrolodione, diketopyrrolopyrroles, benzodithiophene derivatives, and fluorinated heteroarenes with functionalized aryl iodides is proven in solvent-free and non-anhydrous conditions. The reaction is performed in the presence of air and tolerates several functional groups on both the coupling partners, enabling a convenient synthesis of extended heteroaromatic conjugated molecules.

Multiple Routes to Smart Nanostructured Materials from Diatom Microalgae: A Chemical Perspective.

Diatoms are unicellular photosynthetic microalgae, ubiquitously diffused in both marine and freshwater environments, which exist worldwide with more than 100 000 species, each with different morphologies and dimensions, but typically ranging from 10 to 200 µm. A special feature of diatoms is their production of siliceous micro- to nanoporous cell walls, the frustules, whose hierarchical organization of silica layers produces extraordinarily intricate pore patterns. Due to the high surface area, mechanical resistance, unique optical features, and biocompatibility, a number of applications of diatom frustules have been investigated in photonics, sensing, optoelectronics, biomedicine, and energy conversion and storage. Current progress in diatom-based nanotechnology relies primarily on the availability of various strategies to isolate frustules, retaining their morphological features, and modify their chemical composition for applications that are not restricted to those of the bare biosilica produced by diatoms. Chemical or biological methods that decorate, integrate, convert, or mimic diatoms' biosilica shells while preserving their structural features represent powerful tools in developing scalable, low-cost routes to a wide variety of nanostructured smart materials. Here, the different approaches to chemical modification as the basis for the description of applications relating to the different materials thus obtained are presented.

Pd-Catalyzed Thiophene-Aryl Coupling Reaction via C-H Bond Activation in Deep Eutectic Solvents.

Direct arylation of 5-octylthieno[3,4-c]pyrrole-4,6-dione with a series of functionalized aryl iodides via C-H bond activation is demonstrated in a deep eutectic solvent made of choline chloride and urea in non-anhydrous conditions and without exclusion of air. This is the first demonstration of a thiophene-aryl coupling via direct arylation in deep eutectic solvents.

Synthetic Antenna Functioning As Light Harvester in the Whole Visible Region for Enhanced Hybrid Photosynthetic Reaction Centers.

The photosynthetic reaction center (RC) from the Rhodobacter sphaeroides bacterium has been covalently bioconjugated with a NIR-emitting fluorophore (AE800) whose synthesis was specifically tailored to act as artificial antenna harvesting light in the entire visible region. AE800 has a broad absorption spectrum with peaks centered in the absorption gaps of the RC and its emission overlaps the most intense RC absorption bands, ensuring a consistent increase of the protein optical cross section. The covalent hybrid AE800-RC is stable and fully functional. The energy collected by the artificial antenna is transferred to the protein via FRET mechanism, and the hybrid system outperforms by a noteworthy 30% the overall photochemical activity of the native protein under the entire range of visible light. This improvement in the optical characteristic of the photoenzyme demonstrates the effectiveness of the bioconjugation approach as a suitable route to new biohybrid materials for energy conversion, photocatalysis, and biosensing.

Data from two different culture conditions of Thalassiosira weissflogii diatom and from cleaning procedures for obtaining monodisperse nanostructured biosilica.

Diatoms microalgae produce biosilica nanoporous rigid outershells called frustules that exhibit an intricate nanostructured pore pattern. In this paper two specific Thalassiosira weissflogii culture conditions and size control procedures during the diatoms growth are described. Data from white field and fluorescence microscopy, evaluation of cell densities and cell parameters (k value and R value) according to cell culture conditions are listed. Different cleaning procedures for obtaining bare frustules are described. In addition, FTIR and spectrofluorimetric analyses of cleaned biosilica are shown. The data are related to the research article "Chemically Modified Diatoms Biosilica for Bone Cell Growth with Combined Drug-Delivery and Antioxidant Properties" [1].

Biosilica from Living Diatoms: Investigations on Biocompatibility of Bare and Chemically Modified Thalassiosira weissflogii Silica Shells.

In the past decade, mesoporous silica nanoparticles (MSNs) with a large surface area and pore volume have attracted considerable attention for their application in drug delivery and biomedicine. Here we propose biosilica from diatoms as an alternative source of mesoporous materials in the field of multifunctional supports for cell growth: the biosilica surfaces were chemically modified by traditional silanization methods resulting in diatom silica microparticles functionalized with 3-mercaptopropyl-trimethoxysilane (MPTMS) and 3-aminopropyl-triethoxysilane (APTES). Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy analyses revealed that the -SH or -NH2 were successfully grafted onto the biosilica surface. The relationship among the type of functional groups and the cell viability was established as well as the interaction of the cells with the nanoporosity of frustules. These results show that diatom microparticles are promising natural biomaterials suitable for cell growth, and that the surfaces, owing to the mercapto groups, exhibit good biocompatibility.

Chemically Modified Diatoms Biosilica for Bone Cell Growth with Combined Drug-Delivery and Antioxidant Properties.

This month's cover is dedicated to the joint project coordinated by Prof. Gianluca M. Farinola involving the Università degli Studi di Bari Aldo Moro, CNR ICCOM and IMIP in Bari, Università Politecnica delle Marche, and Jaber Innovation. The cover picture shows an SEM image of nanostructured biosilica produced by- Thalassiosira weissflogii- diatoms covalently functionalized with the radical scavenger TEMPO (green disks) and loaded with the antibiotic Ciprofloxacin (red/white capsules), which is used to combat infections related to orthopedic implants. Read the full text of the article at 10.1002/cplu.201402398.

Chemically Modified Diatoms Biosilica for Bone Cell Growth with Combined Drug-Delivery and Antioxidant Properties.

Nanostructured biosilica produced by Thalassiosira weissflogii diatoms is covalently functionalized with the cyclic nitroxide 2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), an efficient scavenger of reactive oxygen species (ROS) in biological systems. Drug delivery properties of the TEMPO-functionalized biosilica are studied for Ciprofloxacin, an antimicrobial thoroughly employed in orthopedic or dental implant related infections. The resulting TEMPO-biosilica, combining Ciprofloxacin drug delivery with anti-oxidant properties, is demonstrated to be a suitable material for fibroblasts and osteoblast-like cells growth.