Comparison of Synthetic Pathways for Obtaining Fluorescent Nanomaterials Based on Halloysite and Carbon Dots for Potential Biological Sensing.

Recently, fluorescent sensors have gained considerable attention due to their high sensitivity, low cost and noninvasiveness. Among the different materials that can be used for this purpose, carbon dots (CDs) represent valuable candidates for applications in sensing. These, indeed, are easily synthesized, show high quantum yield and are highly biocompatible. However, it was pointed out that the photoluminescence properties of these nanomaterials are strictly dependent on the synthetic and purification methods adopted. The presence of halloysite nanotubes (HNTs), a natural, low cost and biocompatible clay mineral, has been found to be efficient in obtaining small and highly monodispersed CDs without long and tedious purification techniques. Herein, we report the comparison of synthetic pathways for obtaining halloysite-N-doped CDs (HNTs-NCDs) that could be used in biological sensing. One was based on the synthesis of N-doped CDs by a bottom-up approach on HNTs' surface by a MW pyrolysis process; the other one was based on the post-modification of pristine N-doped CDs with halloysite derivatives. The evaluation of the best synthetic route was performed by different physico-chemical techniques. It was found that the bottom-up approach led to the formation of N-doped CDs with different functional groups onto the HNTs' surface. This evidence was also translated in the different fluorescence quantum yields and the existence of several functional groups in the obtained materials was investigated by potentiometric titrations. Furthermore, the ability of the synthesized nanomaterials as sensors for Fe3+ ions detection was assessed by spectroscopic measurements, and the cellular uptake was verified by confocal/fluorescence microscopies as well.

Past, Present and Future Perspectives on Halloysite Clay Minerals.

Halloysite nanotubes (HNTs), clay minerals belonging to the kaolin groups, are emerging nanomaterials which have attracted the attention of the scientific community due to their interesting features, such as low-cost, availability and biocompatibility. In addition, their large surface area and tubular structure have led to HNTs' application in different industrial purposes. This review reports a comprehensive overview of the historical background of HNT utilization in the last 20 years. In particular it will focus on the functionalization of the surfaces, both supramolecular and covalent, following applications in several fields, including biomedicine, environmental science and catalysis.

Binding abilities of polyaminocyclodextrins: polarimetric investigations and biological assays.

Three polyaminocyclodextrin materials, obtained by direct reaction between heptakis(6-deoxy-6-iodo)-ß-cyclodextrin and the proper linear polyamines, were investigated for their binding properties, in order to assess their potential applications in biological systems, such as vectors for simultaneous drug and gene cellular uptake or alternatively for the protection of macromolecules. In particular, we exploited polarimetry to test their interaction with some model p-nitroaniline derivatives, chosen as probe guests. The data obtained indicate that binding inside the host cavity is mainly affected by interplay between Coulomb interactions and conformational restraints. Moreover, simultaneous interaction of the cationic polyamine pendant bush at the primary rim was positively assessed. Insights on quantitative aspects of the interaction between our materials and polyanions were investigated by studying the binding with sodium alginate. Finally, the complexation abilities of the same materials towards polynucleotides were assessed by studying their interaction with the model plasmid pUC19. Our results positively highlight the ability of our materials to exploit both the cavity and the polycationic branches, thus functioning as bimodal ligands.

Micellization properties of cardanol as a renewable co-surfactant.

With the aim to improve the features of surfactant solutions in terms of sustainability and renewability we propose the use of hydrogenated natural and sustainable plant-derived cardanol as an additive to commercial surfactants. In the present study we demonstrated that its addition, in amounts as high as 10%, to commercial surfactants of different charge does not significantly affect surfactant properties. Conversely, the presence of hydrogenated cardanol can strongly affect spectrophotometric determination of CMC if preferential interactions with the dyes used take place. This latter evidence may be profitably exploited in surfactant manufacturing by considering that the concurrent presence of a rigid organic molecule such as Orange OT and 10% hydrogenated cardanol decreases the CMC of CTAB up to 65 times.

Development of alginate film filled with halloysite-carbon dots for active food packaging.

The development of functional bionanocomposites for active food packaging is of current interest to replace non-biodegradable plastic coatings. In the present work, we report the synthesis of an alginate-based nanocomposite filled with modified halloysite nanotubes (HNTs) to develop coatings with improved barrier properties for food packaging. Firstly, HNTs were chemically modified by the introduction of carbon dots units (CDs) onto their external surface (HNTs-CDs) obtaining a nanomaterial where CDs are uniformly present onto the tubes as verified by morphological investigations, with good UV absorption and antioxidant properties. Afterwards, these were dispersed in the alginate matrix to obtain the alginate/HNTs-CDs nanocomposite (Alg/HNTs-CDs) whose morphology was imaged by AFM measurements. The UV and water barrier properties (in terms of moisture content and water vapor permeability) were investigated, and the antioxidant properties were evaluated as well. To confer some antimicrobial properties to the final nanocomposite, the synthetized filler was loaded with a natural extract (E) from M. cisplatensis. Finally, the extract kinetic release both from the filler and from the nanocomposite was studied in a medium mimicking a food simulant and preliminary studies on the effect of Alg/HNTs-CDs/E on coated and uncoated fruits, specifically apples and bananas were also carried out.

Thixotropic Hydrogels Based on Laponite® and Cucurbituril for Delivery of Lipophilic Drug Molecules.

Invited for this month's cover are the collaborating groups of Prof. Serena Riela at University of Catania, Prof. César Viseras at University of Granada and Dr. Ignacio Sainz-Diaz at Instituto Andaluz de Ciencias de la Tierra. The cover picture shows the possible application of the developed system. In particular, flufenamic acid, anti-inflammatory and anti-pyretic drug, was complexed into cucurbituril cavity and the supramolecular system obtained was used as filler for laponite® hydrogel for its topical delivery. More information can be found in the Research Article by Viseras-Iborra, Riela, and co-workers.

Thixotropic Hydrogels Based on Laponite® and Cucurbituril for Delivery of Lipophilic Drug Molecules.

Nowadays the use of hydrogels for biomedical purposes is increasing because of their interesting features that allow the development of targeted drug delivery systems. Herein, hydrogel based on Laponite® (Lap) clay mineral as gelator and cucurbit[6]uril (CB[6]) molecules were synthetized for the delivery of flufenamic acid (FFA) for potential topical application. Firstly, the interaction between CB[6] and FFA was assessed by UV-vis spectroscopic measurements and molecular modeling calculations. Then, the obtained complex was used as filler for Lap hydrogel (Lap/CB[6]/FFA). The properties of the hydrogel in terms of viscosity and, self-repair abilities were investigated; its morphology was imaged by scanning electron and polarized optical microscopies. Furthermore, the changes in the hydrodynamic radii and in the colloidal stability of CB[6]/Lap mixture were investigated in terms of translational diffusion from dynamic light scattering and ζ-potential measurements. Finally, the kinetic in vitro release of FFA, from Lap/CB[6]/FFA hydrogel, was studied in a medium mimicking the pH of skin and the obtained results were discussed both by an experimental point of view and by molecular modeling calculations.

Hectorite/Phenanthroline-Based Nanomaterial as Fluorescent Sensor for Zn Ion Detection: A Theoretical and Experimental Study.

The development of fluorescent materials that can act as sensors for the determination of metal ions in biological fluids is important since they show, among others, high sensitivity and specificity. However, most of the molecules that are used for these purposes possess a very low solubility in aqueous media, and, thus, it is necessary to adopt some derivation strategies. Clay minerals, for example, hectorite, as natural materials, are biocompatible and available in large amounts at a very low cost that have been extensively used as carrier systems for the delivery of different hydrophobic species. In the present work, we report the synthesis and characterization of a hectorite/phenanthroline nanomaterial as a potential fluorescent sensor for Zn ion detection in water. The interaction of phenanthroline with the Ht interlaminar space was thoroughly investigated, via both theoretical and experimental studies (i.e., thermogravimetry, FT-IR, UV-vis and fluorescence spectroscopies and XRD measurements), while its morphology was imaged by scanning electron microscopy. Afterwards, the possibility to use it as sensor for the detection of Zn2+ ions, in comparison to other metal ions, was investigated through fluorescent measurements, and the stability of the solid Ht/Phe/Zn complex was assessed by different experimental and theoretical measurements.

Carrier capability of halloysite nanotubes for the intracellular delivery of antisense PNA targeting mRNA of neuroglobin gene.

Peptide nucleic acid (PNA) is a DNA mimic that shows good stability against nucleases and proteases, forming strongly recognized complementary strands of DNA and RNA. However, due to its feeble ability to cross the cellular membrane, PNA activity and its targeting gene action is limited. Halloysite nanotubes (HNTs) are a natural and low-cost aluminosilicate clay. Because of their peculiar ability to cross cellular membrane, HNTs represent a valuable candidate for delivering genetic materials into cells. Herein, two differently charged 12-mer PNAs capable of recognizing as molecular target a 12-mer DNA molecule mimicking a purine-rich tract of neuroglobin were synthetized and loaded onto HNTs by electrostatic attraction interactions. After characterization, the kinetic release was also assessed in media mimicking physiological conditions. Resonance light scattering measurements assessed their ability to bind complementary single-stranded DNA. Furthermore, their intracellular delivery was assessed by confocal laser scanning microscopy on living MCF-7 cells incubated with fluorescence isothiocyanate (FITC)-PNA and HNTs labeled with a probe. The nanomaterials were found to cross cellular membrane and cell nuclei efficiently. Finally, it is worth mentioning that the HNTs/PNA can reduce the level of neuroglobin gene expression, as shown by reverse transcription-quantitative polymerase chain reaction and western blotting analysis.

Antioxidant Efficacy and "In Vivo" Safety of a Bentonite/Vitamin C Hybrid.

L-ascorbic acid (LAA), commonly known as vitamin C, is an excellent and recognized antioxidant molecule used in pharmaceutical and cosmetic formulations. Several strategies have been developed in order to preserve its chemical stability, connected with its antioxidant power, but there is little research regarding the employment of natural clays as LAA host. A safe bentonite (Bent)-which was verified by in vivo ophthalmic irritability and acute dermal toxicity assays-was used as carrier of LAA. The supramolecular complex between LAA and clay may constitute an excellent alternative, since the molecule integrity does not seem to be affected, at least from the point of view of its antioxidant capacity. The Bent/LAA hybrid was prepared and characterized through ultraviolet (UV) spectroscopy, X-ray diffraction (XRD), infrared (IR) spectroscopy, thermogravimetric analysis (TG/DTG) and zeta potential measurements. Photostability and antioxidant capacity tests were also performed. The LAA incorporation into Bent clay was demonstrated, as well as the drug stability due to the Bent photoprotective effect onto the LAA molecule. Moreover, the antioxidant capacity of the drug in the Bent/LAA composite was confirmed.

Nanoformulations based on collagenases loaded into halloysite/Veegum® clay minerals for potential pharmaceutical applications.

The design and development of nanomaterials capable of penetrate cancer cells is fundamental when anticancer therapy is involved. The use of collagenase (Col) is useful since this enzyme can degrade collagen, mainly present in the tumor extracellular matrix. However, its use is often limited since collagenase suffers from inactivation and short half-life. Use of recombinant ultrapure collagenase or carrier systems for their delivery are among the strategies adopted to increase the enzyme stability. Herein, based on the more stability showed by recombinant enzymes and the possibility to use them in anticancer therapy, we propose a novel strategy to further increase their stability by using halloysite nanotubes (HNTs) as carrier. ColG and ColH were supramolecularly loaded onto HNTs and used as fillers for Veegum gels. The systems could be used for potential local administration of collagenases for solid tumor treatment. All techniques adopted for characterization showed that halloysite interacts with collagenases in different ways depending with the Col considered. Furthermore, the hydrogels showed a very slow release of the collagenases within 24 h. Finally, biological assays were performed by studying the digestion of a type-I collagen matrix highlighting that once released the Col still possessed some activity. Thus we developed carrier systems that could further increase the high recombinant collagenases stability, preventing their inactivation in future in vivo applications for potential local tumor treatment.

Modification of halloysite lumen with dopamine derivatives as filler for antibiofilm coating.

HYPOTHESIS: Development of nanocomposite coating with antibiofilm properties is of fundamental importance to efficient fight biofilm formation preventing infections in biomedical area. In this context, halloysite nanotubes (HNTs), biocompatible and low-cost clay mineral, have been efficiently used as filler for different polymeric matrices affording several nanocomposites with appealing antimicrobial properties. The modification of HNTs surfaces represents a valuable strategy to improve the utilization of the clay for biological purposes. EXPERIMENTS: Herein, the covalent modification of the HNTs lumen with properly designed dopamine derivatives with different perfluoroalkyl chain length is reported. The obtained nanomaterials are thoroughly characterized by several techniques. As proof of concept the antibiofilm properties on E. coli strain of the nanomaterials are assayed as well. Finally, the HNTs fillers were introduced into a polydopamine matrix allowing for the preparation of functional coatings, resistant to formation of microbial biofilms. FINDINGS: All characterization methods proved the selectivity of the modification and the increased hydrophobicity of the lumen. In particular 27Al solid state nuclear magnetic resonance (NMR) spectra showed a upfield shift of the Al signal. Studies on the antibiofilm properties highlighted different activities according to the length of perfluoroalkyl chains of organic molecules as proved by 19F solid state NMR spectra. The synthetized materials were promising for future application as coatings on medical implants.

Exploiting the interaction between halloysite and charged PNAs for their controlled release.

The design and development of nanomaterials that could be used in nanomedicine are of fundamental importance to obtain smart nanosystems for the treatment of several diseases. Halloysite, because of its interesting features, represents a suitable nanomaterial for the delivery of different biologically active species. Among them, peptide nucleic acids (PNAs) have attracted considerable attention in recent decades for their potential applications in both molecular antisense diagnosis and as therapeutic agents, although up to now, the actual clinical applications have been very limited. Herein we report a systematic study on the supramolecular interaction of three differently charged PNAs with halloysite. Understanding the interaction mode of charged molecules with the clay surfaces represents a key factor for the future design and development of halloysite based materials which could be used for the delivery and subsequent intracellular release of PNA molecules. Thus, three different PNA tetramers, chosen as models, were synthesized and loaded onto the clay. The obtained nanomaterials were characterized using spectroscopic studies and thermogravimetric analysis, and their morphologies were studied using high angle annular dark field transmission electron microscopy (HAADF/STEM) coupled with Energy Dispersive X-ray spectroscopy (EDX). The aqueous mobility of the three different nanomaterials was investigated by dynamic light scattering (DLS) and ζ-potential measurements. The release of PNA tetramers from the nanomaterials was investigated at two different pH values, mimicking physiological conditions. Finally, to better understand the stability of the synthesized PNAs and their interactions with HNTs, molecular modelling calculations were also performed. The obtained results showed that PNA tetramers interact in different ways with HNT surfaces according to their charge which influences their kinetic release in media mimicking physiological conditions.

Antimicrobial Nanomaterials Based on Halloysite Clay Mineral: Research Advances and Outlook.

Bacterial infections represent one of the major causes of mortality worldwide. Therefore, over the years, several nanomaterials with antibacterial properties have been developed. In this context, clay minerals, because of their intrinsic properties, have been efficiently used as antimicrobial agents since ancient times. Halloysite nanotubes are one of the emerging nanomaterials that have found application as antimicrobial agents in several fields. In this review, we summarize some examples of the use of pristine and modified halloysite nanotubes as antimicrobial agents, scaffolds for wound healing and orthopedic implants, fillers for active food packaging, and carriers for pesticides in food pest control.

Prodrug based on halloysite delivery systems to improve the antitumor ability of methotrexate in leukemia cell lines.

The prodrug approach, as well as the development of specific systems able to deliver a chemotherapeutic agent in the target site, decreasing the side effects often associated with its administration, are still a challenging. In this context, both methotrexate drug molecules (MTX) and biotin ligand moieties, whose receptors are overexpressed on the surface of several cancer cells, were loaded on halloysite nanotubes (HNTs) to develop nanomaterial based on multifunctional and "smart" delivery systems. To highlight the crucial role played by biotin, carrier systems based on HNTs and MTX were also synthetized. In detail, several approaches were envisaged: i) a supramolecular interaction between the clay and the drug; ii) a covalent grafting of the drug onto the HNTs external surface and, iii) a combination of both approaches. The nanomaterials obtained were characterized by thermogravimetric analysis, FT-IR, and UV-vis spectroscopies, DLS and ζ-potential measurements and the morphologies were imaged by HAADF/STEM investigations. Kinetic release experiments at different pH conditions were also performed. Finally, as a proof-of-concept application of our pro-drug delivery systems based on HNTs in cancer therapy, the cytotoxic effects were evaluated on acute myeloid leukemia cell lines, HL60 and its multidrug resistance variant, HL60R. The obtained results showed that both the MTX prodrug system and the biotinylated ones played a crucial role in the biological activity and, they are promising agents for the cancer treatments.

Exploring the cellular uptake of hectorite clay mineral and its drug carrier capabilities.

In the last years, the use of clay minerals for pharmaceutical purposes has increased due to their interesting properties. Hectorite (Ht) is a clay belonging to the smectite group which has attracted attention for applications in biology, tissue engineering and as drug carrier and delivery system. However, the mechanisms involved in Ht cellular uptake and transport into cells, are still unclear. Herein, we used a labeled Ht (Ht/1Cl) to study both the cellular uptake, by confocal laser scanning microscopy, and internalization pathways involved in the cellular uptake, by various endocytosis-inhibiting studies and fluorescence microscopy. These studies highlighted that Ht can penetrate the cellular membrane, localizing mainly in the cytoplasm. The main intracellular transport mechanisms are the ATP-dependent ones and those where filaments and microtubules are involved. Finally, as proof of concept for the potential of Ht as carrier system, we envisaged the covalent grafting of the anticancer molecule methotrexate (MTX), chosen as model, to obtain the Ht-MTX nanomaterial. The covalent linkage was confirmed by several techniques and the morphology of the obtained nanomaterial was imaged by SEM and TEM investigations. The kinetic release of the drug from the Ht-MTX nanomaterial in physiological conditions was studied as well. Furthermore, cytotoxic studies on different cell lines, namely, HL-60, HL-60R, MCF-7, 5637, UMUC3 and RT112 showed that Ht could be a promising material for anticancer therapy.

Nanocarrier based on halloysite and fluorescent probe for intracellular delivery of peptide nucleic acids.

The development of systems able to deliver genetic material into a target site is a challenge for modern medicine. Single-stranded peptide nucleic acids have attracted attention as promising therapeutic molecules for diagnostic and gene therapy. However, their poor cell membrane permeability represents a drawback for biomedical applications. Halloysite nanotubes (HNTs) are emerging materials in drug delivery applications both for their ability to penetrate cell membranes and for enhancing the solubility of drugs in biological media. Herein, we report the first example of the use of a nanocarrier based on halloysite labelled with fluorescent switchable halochromic oxazine molecules, to deliver a single-stranded peptide nucleic acids tetramer (PNAts) into living cells. The PNAts is covalently attached to halloysite (HNTs-PNA), whereas the fluorescent probe supramolecularly interacts with HNTs. The ability of the nanomaterial to bind complementary single-stranded DNA was assessed by resonance light scattering measurements. Finally, studies of cellular uptake were carried out by confocal laser scanning microscopy on normal and tumoral cell lines. This work highlights the usefulness of the covalent approach to generate HNTs-PNA nanomaterials for the potential targeting of future specific nucleic acids in living cells, which could open the doorway to novel possibilities for theranostic and gene therapy applications.

Site-specific halloysite functionalization by polydopamine: A new synthetic route for potential near infrared-activated delivery system.

Halloysite nanotubes (HNTs) represent a versatile core structure for the design of functional nanosystems of biomedical interest. However, the development of selective methodologies for the site-controlled functionalization of the nanotubes at specific sites is not an easy task. This study aims to accomplish a procedure for the site-selective/specific, "pin-point", functionalization of HNTs with polydopamine (HNTs@PDA). This goal was achieved, at pH 6.5, by exploiting the basicity of ZnO nanoparticles anchored on the HNTs external surface (HNTs@ZnO) to induce a punctual polydopamine polymerization and coating. The morphology and the chemical composition of the nanomaterial was demonstrated by several techniques. Turbidimetric analysis showed that PDA coating affected the aqueous stability of HNTs@PDA compared to both HNTs@ZnO and HNTs. Notably, hyperthermia studies revealed that the nanomaterial induced a local thermic rise, up to 50 °C, under near-infrared (NIR) irradiation. Furthermore, secondary functionalization of HNTs@PDA by selective grafting of biotin onto the PDA coating followed by avidin binding was also accomplished.

A study on the essential oil of Ferulago campestris: how much does extraction method influence the oil composition?

The essential oil of different parts of Ferulago campestris (Bess.) collected in Sicily has been extracted by microwave-assisted hydrodistillation (MAHD) and by classic hydrodistillation (HD). A comparative qualitative-quantitative study on the composition of the oils was carried out. A total of 100 compounds were identified in the oils obtained by MAHD, whereas 88 compounds characterized the HD oils. The most prominent components were, in all different parts of F. campestris and in both extraction methods, 2,4,5-trimethylbenzaldehyde and 2,4,6-trimethylbenzaldehyde isomers; the latter was not previously found. The attempt to evaluate where the oil components are located in all parts of the plant was carried out by means of a kinetic study. Then, electron microscopy observation on the different parts before and after MAHD and HD was performed.

Study of Uptake Mechanisms of Halloysite Nanotubes in Different Cell Lines.

PURPOSE: Halloysite nanotubes (HNTs) are a natural aluminosilicate clay with a chemical formula of Al2Si2O5(OH)4×nH2O and a hollow tubular structure. Due to their peculiar structure, HNTs can play an important role as a drug carrier system. Currently, the mechanism by which HNTs are internalized into living cells, and what is the transport pathway, is still unclear. Therefore, this study aimed at establishing the in vitro mechanism by which halloysite nanotubes could be internalized, using phagocytic and non-phagocytic cell lines as models. METHODS: The HNT/CURBO hybrid system, where a fluorescent probe (CURBO) is confined in the HNT lumen, has been used as a model to study the transport pathway mechanisms of HNTs. The cytocompatibility of HNT/CURBO on cell lines model was investigated by MTS assay. In order to identify the internalization pathway involved in the cellular uptake, we performed various endocytosis-inhibiting studies, and we used fluorescence microscopy to verify the nanomaterial internalization by cells. We evaluated the haemolytic effect of HNT/CURBO placed in contact with human red blood cells (HRBCs), by reading the absorbance value of the supernatant at 570 nm. RESULTS: The HNT/CURBO is highly biocompatible and does not have an appreciable haemolytic effect. The results of the inhibition tests have shown that the internalization process of nanotubes occurs in an energy-dependent manner in both the investigated cell lines, although they have different characteristics. In particular, in non-phagocytic cells, clathrin-dependent and independent endocytosis are involved. In phagocytic cells, in addition to phagocytosis and clathrin-dependent endocytosis, microtubules also participate in the halloysite cellular trafficking. Upon internalization by cells, HNT/CURBO is localized in the cytoplasmic area, particularly in the perinuclear region. CONCLUSION: Understanding the cellular transport pathways of HNTs can help in the rational design of novel drug delivery systems and can be of great value for their applications in biotechnology.