Diatom-Based Nanomedicine for Colorectal Cancer Treatment: New Approaches for Old Challenges.

Colorectal cancer is among the most prevalent and lethal cancers globally. To address this emergency, countries have developed diffuse screening programs and innovative surgical techniques with a consequent decrease in mortality rates in non-metastatic patients. However, five years after diagnosis, metastatic CRC is still characterized by less than 20% survival. Most patients with metastatic CRC cannot be surgically treated. For them, the only option is treatment with conventional chemotherapies, which cause harmful side effects in normal tissues. In this context, nanomedicine can help traditional medicine overcome its limits. Diatomite nanoparticles (DNPs) are innovative nano-based drug delivery systems derived from the powder of diatom shells. Diatomite is a porous biosilica largely found in many areas of the world and approved by the Food and Drug Administration (FDA) for pharmaceutical and animal feed formulations. Diatomite nanoparticles with a size between 300 and 400 nm were shown to be biocompatible nanocarriers capable of delivering chemotherapeutic agents against specific targets while reducing off-target effects. This review discusses the treatment of colorectal cancer with conventional methods, highlighting the drawbacks of standard medicine and exploring innovative options based on the use of diatomite-based drug delivery systems. Three targeted treatments are considered: anti-angiogenetic drugs, antimetastatic drugs, and immune checkpoint inhibitors.

microRNA Detection via Nanostructured Biochips for Early Cancer Diagnostics.

MicroRNA (miRNA) are constituted of approximately 22 nucleotides and play an important role in the regulation of many physiological functions and diseases. In the last 10 years, an increasing interest has been recorded in studying the expression profile of miRNAs in cancer. Real time-quantitative polymerase chain reaction (RT-qPCR), microarrays, and small RNA sequencing represent the gold standard techniques used in the last 30 years as detection methods. The advent of nanotechnology has allowed the fabrication of nanostructured biosensors which are widely exploited in the diagnostic field. Nanostructured biosensors offer many advantages: (i) their small size allows the construction of portable, wearable, and low-cost products; (ii) the large surface-volume ratio enables the loading of a great number of biorecognition elements (e.g., probes, receptors); and (iii) direct contact of the recognition element with the analyte increases the sensitivity and specificity inducing low limits of detection (LOD). In this review, the role of nanostructured biosensors in miRNA detection is explored, focusing on electrochemical and optical sensing. In particular, four types of nanomaterials (metallic nanoparticles, graphene oxide, quantum dots, and nanostructured polymers) are reported for both detection strategies with the aim to show their distinct properties and applications.

ZnO Tetrapods for Label-Free Optical Biosensing: Physicochemical Characterization and Functionalization Strategies.

In this study, we fabricated three different ZnO tetrapodal nanostructures (ZnO-Ts) by a combustion process and studied their physicochemical properties by different techniques to evaluate their potentiality for label-free biosensing purposes. Then, we explored the chemical reactivity of ZnO-Ts by quantifying the available functional hydroxyl groups (-OH) on the transducer surface necessary for biosensor development. The best ZnO-T sample was chemically modified and bioconjugated with biotin as a model bioprobe by a multi-step procedure based on silanization and carbodiimide chemistry. The results demonstrated that the ZnO-Ts could be easily and efficiently biomodified, and sensing experiments based on the streptavidin target detection confirmed these structures' suitability for biosensing applications.

Development of Surface Chemical Strategies for Synthesizing Redox-Responsive Diatomite Nanoparticles as a Green Platform for On-Demand Intracellular Release of an Antisense Peptide Nucleic Acid Anticancer Agent.

Redox-responsive silica drug delivery systems are synthesized by aeco-friendly diatomite source to achieve on-demand release of peptide nucleic acid (PNA) in tumor reducing microenvironment, aiming to inhibit the immune checkpoint programmed cell death 1 receptor/programmed cell death receptor ligand 1 (PD-1/PD-L1) in cancer cells. The nanoparticles (NPs) are coated with polyethylene glycol chains as gatekeepers to improve their physicochemical properties and control drug release through the cleavable disulfide bonds (S-S) in a reductive environment. This study describes different chemical conditions to achieve the highest NPs' surface functionalization yield, exploring both multistep and one-pot chemical functionalization strategies. The best formulation is used for covalent PNA conjugation via the S-S bond reaching a loading degree of 306 ± 25 µg PNA mg-1 DNPs . These systems are used for in vitro studies to evaluate the kinetic release, biocompatibility, cellular uptake, and activity on different cancer cells expressing high levels of PD-L1. The obtained results prove the safety of the NPs up to 200 µg mL-1 and their advantage for controlling and enhancing the PNA intracellular release as well as antitumor activity. Moreover, the downregulation of PD-L1 observed only with MDA-MB-231 cancer cells paves the way for targeted immunotherapy.

SERS Quantification of Galunisertib Delivery in Colorectal Cancer Cells by Plasmonic-Assisted Diatomite Nanoparticles.

The small molecule Galunisertib (LY2157299, LY) shows multiple anticancer activities blocking the transforming growth factor-ß1 receptor, responsible for the epithelial-to-mesenchymal transition (EMT) by which colorectal cancer (CRC) cells acquire migratory and metastatic capacities. However, frequent dosing of LY can produce highly toxic metabolites. Alternative strategies to reduce drug side effects can rely on nanoscale drug delivery systems that have led to a medical revolution in the treatment of cancer, improving drug efficacy and lowering drug toxicity. Here, a hybrid nanosystem (DNP-AuNPs-LY@Gel) made of a porous diatomite nanoparticle decorated with plasmonic gold nanoparticles, in which LY is retained by a gelatin shell, is proposed. The multifunctional capability of the nanosystem is demonstrated by investigating the efficient LY delivery, the enhanced EMT reversion in CRCs and the intracellular quantification of drug release with a sub-femtogram resolution by surface-enhanced Raman spectroscopy (SERS). The LY release trigger is the pH sensitivity of the gelatin shell to the CRC acidic microenvironment. The drug release is real-time monitored at single-cell level by analyzing the SERS signals of LY in CRC cells. The higher efficiency of LY delivered by the DNP-AuNPs-LY@Gel complex paves the way to an alternative strategy for lowering drug dosing and consequent side effects.

Porous Silicon Optical Devices: Recent Advances in Biosensing Applications.

This review summarizes the leading advancements in porous silicon (PSi) optical-biosensors, achieved over the past five years. The cost-effective fabrication process, the high internal surface area, the tunable pore size, and the photonic properties made the PSi an appealing transducing substrate for biosensing purposes, with applications in different research fields. Different optical PSi biosensors are reviewed and classified into four classes, based on the different biorecognition elements immobilized on the surface of the transducing material. The PL signal modulation and the effective refractive index changes of the porous matrix are the main optical transduction mechanisms discussed herein. The approaches that are commonly employed to chemically stabilize and functionalize the PSi surface are described.

Design of Gelatin-Capped Plasmonic-Diatomite Nanoparticles with Enhanced Galunisertib Loading Capacity for Drug Delivery Applications.

Inorganic diatomite nanoparticles (DNPs) have gained increasing interest as drug delivery systems due to their porous structure, long half-life, thermal and chemical stability. Gold nanoparticles (AuNPs) provide DNPs with intriguing optical features that can be engineered and optimized for sensing and drug delivery applications. In this work, we combine DNPs with gelatin stabilized AuNPs for the development of an optical platform for Galunisertib delivery. To improve the DNP loading capacity, the hybrid platform is capped with gelatin shells of increasing thicknesses. Here, for the first time, full optical modeling of the hybrid system is proposed to monitor both the gelatin generation, degradation, and consequent Galunisertib release by simple spectroscopic measurements. Indeed, the shell thickness is optically estimated as a function of the polymer concentration by exploiting the localized surface plasmon resonance shifts of AuNPs. We simultaneously prove the enhancement of the drug loading capacity of DNPs and that the theoretical modeling represents an efficient predictive tool to design polymer-coated nanocarriers.

Recent Advances on Diatom-Based Biosensors.

Porous materials showing some useful transducing features, i.e., any changes in their physical or chemical properties as a consequence of molecular interaction, are very attractive in the realization of sensors and biosensors. Diatom frustules have been gaining support for biosensors since they are made of nanostructured amorphous silica, but do not require any nano-fabrication step; their surface can be easily functionalized and customized for specific application; diatom frustules are photoluminescent, and they can be found in almost every pond of water on the Earth, thus assuring large and low-cost availability. In this review, the most recent advances in diatom-based biosensors are reported, and a perspective view on future developments is given.

Functionalized Polymeric Materials with Bio-Derived Antimicrobial Peptides for "Active" Packaging.

Food packaging is not only a simple protective barrier, but a real "active" component, which is expected to preserve food quality, safety and shelf-life. Therefore, the materials used for packaging production should show peculiar features and properties. Specifically, antimicrobial packaging has recently gained great attention with respect to both social and economic impacts. In this paper, the results obtained by using a polymer material functionalized by a small synthetic peptide as "active" packaging are reported. The surface of Polyethylene Terephthalate (PET), one of the most commonly used plastic materials in food packaging, was plasma-activated and covalently bio-conjugated to a bactenecin-derivative peptide named 1018K6, previously characterized in terms of antimicrobial and antibiofilm activities. The immobilization of the peptide occurred at a high yield and no release was observed under different environmental conditions. Moreover, preliminary data clearly demonstrated that the "active" packaging was able to significantly reduce the total bacterial count together with yeast and mold spoilage in food-dairy products. Finally, the functionalized-PET polymer showed stronger efficiency in inhibiting biofilm growth, using a Listeria monocytogenes strain isolated from food products. The use of these "active" materials would greatly decrease the risk of pathogen development and increase the shelf-life in the food industry, showing a real potential against a panel of microorganisms upon exposure to fresh and stored products, high chemical stability and re-use possibility.

Porous Silicon-Based Aptasensors: The Next Generation of Label-Free Devices for Health Monitoring.

Aptamers are artificial nucleic acid ligands identified and obtained from combinatorial libraries of synthetic nucleic acids through the in vitro process SELEX (systematic evolution of ligands by exponential enrichment). Aptamers are able to bind an ample range of non-nucleic acid targets with great specificity and affinity. Devices based on aptamers as bio-recognition elements open up a new generation of biosensors called aptasensors. This review focuses on some recent achievements in the design of advanced label-free optical aptasensors using porous silicon (PSi) as a transducer surface for the detection of pathogenic microorganisms and diagnostic molecules with high sensitivity, reliability and low limit of detection (LoD).

Small Synthetic Peptides Bioconjugated to Hybrid Gold Nanoparticles Destroy Potentially Deadly Bacteria at Submicromolar Concentrations.

Synthetic antibacterial peptides are advanced weapons that scientists design and produce to confront current threats of harmful and mortal pathogens, which could affect humans in everyday life. Recently, many small amino acid sequences, greatly efficient in their antibacterial action, have been reported in the literature. To date, only a few synthetic peptides, acting at micromolar or even tenths of micromolar concentrations, are on the market as commercial products, mainly because of their high cost of production. In this context, materials science can provide fundamental help by engineering small synthetic peptides, powered by hybrid gold nanoparticles, which have been found to strongly enhance antimicrobial activity against bacterial infections. Submicromolar concentrations of the 1018K6 peptide, bioconjugated to hybrid polymer-gold nanoparticles, kill almost 100% of pathogen bacteria, such as Listeria and Salmonella genera, paving the way for economically sustainable commercial products based on this synthetic nanocomplex.

Gold decorated porous biosilica nanodevices for advanced medicine.

Diatomite is a fossil material made of amorphous porous silica. In this work, polyethylene glycol (PEG)-modified diatomite NPs (PEG-DNPs) are decorated with gold NPs (AuNPs) by one-pot liquid-phase synthesis. Nanocomplexes (PEG-DNPs@AuNPs), with an average size of about 450 nm, are characterized by dynamic light scattering, electron microscopy, nitrogen adsorption/desorption analysis, UV-vis and photoluminescence spectroscopies. Preliminary studies on the use of the nanocomplex in nanomedicine are also presented. Tests performed incubating PEG-DNPs@AuNPs in physiological conditions reveal a good stability of material. Cellular uptake of labeled PEG-DNPs@AuNPs is investigated by confocal microscopy after incubation with human cervix epithelioid carcinoma (HeLa) cells up to 48 h: an efficient cytoplasmic localization is observed. In vitro cytotoxicity of nanocomplexes with a concentration up to 400 µg ml-1 for 72 h is also evaluated. The results suggest the use of PEG-DNPs@AuNPs as advanced nanodevices adding imaging features to the nanocomplexes, due to AuNPs as contrast agent.

Photoluminescence enhancement of graphene oxide emission by infiltration in an aperiodic porous silicon multilayer.

Graphene oxide (GO) is a photoluminescent material whose application in integrated optoelectronics has been strongly limited due to poor emission intensity and handling procedures not compatible with standard microelectronic ones. In this work, a hybrid GO-porous silicon (GO-PSi) structure is realized in order to investigate the emission properties of GO infiltrated into an aperiodic porous multilayered matrix. A photoluminescence enhancement by a factor 32, compared to the same amount of GO deposited on a flat silicon surface, is demonstrated. Photoluminescence measurements also show wavelength modulation of the emitted signal.

One-pot synthesis of a gold nanoparticle-Vmh2 hydrophobin nanobiocomplex for glucose monitoring.

HydrophobinVmh2 is a small amphiphilic protein, which self-assembles on different surfaces and naturally interacts with glucose. Here, we report on the synthesis of a nanobiocomplex made of polyethylene glycol, Vmh2 and gold nanoparticles by a one-step process and on its ability to recognise glucose in an aqueous solution at 0.3-0.6-1.2 mg ml(-1) concentrations. Even though the Vmh2 proteins are intrinsically bonded to the gold core, effective glucose interaction monitoring was demonstrated by using dynamic light scattering, ultraviolet-visible, polarization-modulated infrared reflection-absorption and x-ray photoelectron spectroscopies. Experimental results highlighted an affinity constant of 7.3 ± 0.3 mg ml(-1) between the nanobiosystem and the sugar, and a detection sensitivity of 0.13 ± 0.06 a.u./mg ml(-1).

Diatom Valve Three-Dimensional Representation: A New Imaging Method Based on Combined Microscopies.

The frustule of diatoms, unicellular microalgae, shows very interesting photonic features, generally related to its complicated and quasi-periodic micro- and nano-structure. In order to simulate light propagation inside and through this natural structure, it is important to develop three-dimensional (3D) models for synthetic replica with high spatial resolution. In this paper, we present a new method that generates images of microscopic diatoms with high definition, by merging scanning electron microscopy and digital holography microscopy or atomic force microscopy data. Starting from two digital images, both acquired separately with standard characterization procedures, a high spatial resolution (Δz = λ/20, Δx = Δy ≅ 100 nm, at least) 3D model of the object has been generated. Then, the two sets of data have been processed by matrix formalism, using an original mathematical algorithm implemented on a commercially available software. The developed methodology could be also of broad interest in the design and fabrication of micro-opto-electro-mechanical systems.

Diatomite biosilica nanocarriers for siRNA transport inside cancer cells.

BACKGROUND: Diatomite is a natural porous biomaterial of sedimentary origin, formed by fragments of diatom siliceous skeletons, called "frustules". Due to large availability in many areas of the world, chemical stability, and non-toxicity, these fossil structures have been widespread used in lot of industrial applications, such as food production, water extracting agent, production of cosmetics and pharmaceutics. However, diatomite is surprisingly still rarely used in biomedical applications. In this work, we exploit diatomite nanoparticles for small interfering ribonucleic acid (siRNA) transport inside human epidermoid cancer cells (H1355). METHODS: Morphology and composition of diatomite microfrustules (average size lower than 40µm) are investigated by scanning electron microscopy equipped by energy dispersive X-ray spectroscopy, Fourier transform infrared analysis, and photoluminescence measurements. Nanometric porous particles (average size lower than 450nm) are obtained by mechanical crushing, sonication, and filtering of micrometric frustules. siRNA bioconjugation is performed on both micrometric and nanometric fragments by silanization. RESULTS: In-vitro experiments show very low toxicity on exposure of the cells to diatomite nanoparticle concentration up to 300µg/ml for 72h. Confocal microscopy imaging performed on cancer cells incubated with siRNA conjugated nanoparticles demonstrates a cytoplasmatic localization of vectors. Gene silencing by delivered siRNA is also demonstrated. CONCLUSION: Our studies endorse diatomite nanoparticles as non-toxic nanocarriers for siRNA transport in cancer cells. GENERAL SIGNIFICANCE: siRNA is a powerful molecular tool for cancer treatment but its delivery is inefficient due to the difficulty to penetrate the cell membrane. siRNA-diatomite nanoconjugate may be well suited for delivery of therapeutic to cancer cells.

Hydrophobin-coated plates as matrix-assisted laser desorption/ionization sample support for peptide/protein analysis.

Fungal hydrophobins are amphipathic self-assembling proteins. Vmh2 hydrophobin, prepared from mycelial cultures of the basidiomycete fungus Pleurotus ostreatus, spontaneously forms a stable and homogeneous layer on solid surfaces and is able to strongly absorb proteins even in their active forms. In this work, we have exploited the Vmh2 self-assembled layer as a novel coating of a matrix-assisted laser desorption/ionization (MALDI) steel sample-loading plate. Mixtures of standard proteins, as well as tryptic peptides, in the nanomolar-femtomolar range were analyzed in the presence of salts and denaturants. As evidence on a real complex sample, crude human serum was also analyzed and spectra over a wide mass range were acquired. A comparison of this novel coating method with both standard desalting techniques and recently reported on-plate desalting methods was also performed. The results demonstrate that Vmh2 coating of MALDI plates allows for a very simple and effective desalting method suitable for development of lab-on-a-plate platforms focused on proteomic applications.

Effect of the molecular weight on the sensing mechanism in polyethylene glycol diacrylate/gold nanocomposite optical transducers.

The combination of plasmonic nanoparticles and hydrogels results in nanocomposite materials with unprecedented properties that give rise to powerful platforms for optical biosensing. Herein, we propose a physicochemical characterization of plasmonic hydrogel nanocomposites made of polyethylene glycol diacrylate (PEGDA) hydrogels with increasing molecular weights (700-10000 Da) and gold nanoparticles (AuNPs, ∼60 nm). The swelling capability, mechanical properties, and thermal responses of the nanocomposites are analyzed and the combination with the resulting optical properties is elucidated. The different optomechanical properties of the proposed nanocomposites result in different transduction mechanisms, which can be exploited for several biosensing applications. A correlation between the polymer molecular weight, the effective refractive index of the material, and the optical response is found by combining experimental data and numerical simulations. In particular, the localized surface plasmon resonance (LSPR) position of the AuNPs was found to follow a parabolic profile as a function of the monomer molecular weight (MW), while its absorbance intensity was found as inversely proportional to the monomer MW. Low MW PEGDA nanocomposites were found to be responsive to refractive index variations for small molecule sensing. Differently, high MW PEGDA nanocomposites exhibited absorbance intensity increase/decrease as a function of the hydrophobicity/hydrophilicity of the targeted small molecule. The proposed optomechanical model paves the way to the design of innovative platforms for real-life applications, such as wearable sensing, point-of-care testing, and food monitoring via smart packaging devices.

Multiple-pathways light modulation in Pleurosigma strigosum bi-raphid diatom.

Ordered, quasi-ordered, and even disordered nanostructures can be identified as constituent components of several protists, plants and animals, making possible an efficient manipulation of light for intra- and inter- species communication, camouflage, or for the enhancement of primary production. Diatoms are ubiquitous unicellular microalgae inhabiting all the aquatic environments on Earth. They developed, through tens of millions of years of evolution, ultrastructured silica cell walls, the frustules, able to handle optical radiation through multiple diffractive, refractive, and wave-guiding processes, possibly at the basis of their high photosynthetic efficiency. In this study, we employed a range of imaging, spectroscopic and numerical techniques (including transmission imaging, digital holography, photoluminescence spectroscopy, and numerical simulations based on wide-angle beam propagation method) to identify and describe different mechanisms by which Pleurosigma strigosum frustules can modulate optical radiation of different spectral content. Finally, we correlated the optical response of the frustule to the interaction with light in living, individual cells within their aquatic environment following various irradiation treatments. The obtained results demonstrate the favorable transmission of photosynthetic active radiation inside the cell compared to potentially detrimental ultraviolet radiation.

Higher-order G-quadruplex structures and porphyrin ligands: Towards a non-ambiguous relationship.

Herein, we evaluated the interaction of the tetracationic porphyrin H2TCPPSpm4 with three distinct DNA G-quadruplex (G4) models, i.e., the tetramolecular G4 d(TGGGGT)4 (Q1), the 5'-5' stacked G4-dimer [d(CGGAGGT)4]2 (Q2), and a mixture of 5'-5' stacked G-wires [d(5'-CGGT-3'-3'-GGC-5')4]n (Qn). The combined data obtained from UV-Vis, CD, fluorescence, PAGE, RLS, AFM, NMR, and HPLC-SEC experiments allowed us to shed light on the binding mode of H2TCPPSpm4 with the three G4 models differing for the type and the number of available G4 ending faces, the length of the G4 units, and the number of stacked G4 building blocks. Specifically, we found that H2TCPPSpm4 interacted with the shortest Q1 as an end-stacking ligand, whereas the groove binding mode was ascertained in the case of the Q2 and Qn G4 models. In the case of the interaction with Q1 and Qn, we found that H2TCPPSpm4 induces the formation of supramolecular aggregates at porphyrin/G4 ratios higher than 2:1, whereas no significant aggregation was observed for the interaction with Q2 up to the 5:1 ratio. These results unambiguously demonstrated the suitability of porphyrins for the development of specific G4 ligands or G4-targeting diagnostic probes, being H2TCPPSpm4 capable to distinguish between different G4s.