Tuning Peptide-Based Nanofibers for Achieving Selective Doxorubicin Delivery in Triple-Negative Breast Cancer.

Introduction: The design of delivery tools that efficiently transport drugs into cells remains a major challenge in drug development for most pathological conditions. Triple-negative breast cancer (TNBC) is a very aggressive subtype of breast cancer with poor prognosis and limited effective therapeutic options. Purpose: In TNBC treatment, chemotherapy remains the milestone, and doxorubicin (Dox) represents the first-line systemic treatment; however, its non-selective distribution causes a cascade of side effects. To address these problems, we developed a delivery platform based on the self-assembly of amphiphilic peptides carrying several moieties on their surfaces, aimed at targeting, enhancing penetration, and therapy. Methods: Through a single-step self-assembly process, we used amphiphilic peptides to obtain nanofibers decorated on their surfaces with the selected moieties. The surface of the nanofiber was decorated with a cell-penetrating peptide (gH625), an EGFR-targeting peptide (P22), and Dox bound to the cleavage sequence selectively recognized and cleaved by MMP-9 to obtain on-demand drug release. Detailed physicochemical and cellular analyses were performed. Results: The obtained nanofiber (NF-Dox) had a length of 250 nm and a diameter of 10 nm, and it was stable under dilution, ionic strength, and different pH environments. The biological results showed that the presence of gH625 favored the complete internalization of NF-Dox after 1h in MDA-MB 231 cells, mainly through a translocation mechanism. Interestingly, we observed the absence of toxicity of the carrier (NF) on both healthy cells such as HaCaT and TNBC cancer lines, while a similar antiproliferative effect was observed on TNBC cells after the treatment with the free-Dox at 50 µM and NF-Dox carrying 7.5 µM of Dox. Discussion: We envision that this platform is extremely versatile and can be used to efficiently carry and deliver diverse moieties. The knowledge acquired from this study will provide important guidelines for applications in basic research and biomedicine.

Exosomes multiplex profiling, a promising strategy for early diagnosis of laryngeal cancer.

BACKGROUND: Exosomes are nanosized vesicles released from all cells into surrounding biofluids, including cancer cells, and represent a very promising direction in terms of minimally invasive approaches to early disease detection. They carry tumor-specific biological contents such as DNA, RNA, proteins, lipids, and sugars, as well as surface molecules that are able to pinpoint the cellular source. By the above criteria, exosomes may be stratified according to the presence of tissue and disease-specific signatures and, due to their stability in such biofluids as plasma and serum, they represent an indispensable source of vital clinical insights from liquid biopsies, even at the earliest stages of cancer. Therefore, our work aimed to isolate and characterize LCa patients' derived exosomes from serum by Flow Cytometry in order to define a specific epitope signature exploitable for early diagnosis. METHODS: Circulating exosomes were collected from serum collected from 30 LCa patients and 20 healthy volunteers by the use of antibody affinity method exploiting CD63 specific surface marker. Membrane epitopes were then characterized by Flow cytometry multiplex analysis and compared between LCa Patients and Healthy donors. Clinical data were also matched to obtain statistical correlation. RESULTS: A distinct overexpression of CD1c, CD2, CD3, CD4, CD11c, CD14, CD20, CD44, CD56, CD105, CD146, and CD209 was identified in LCa patients compared to healthy controls, correlating positively with tumor presence. Conversely, CD24, CD31, and CD40, though not overexpressed in tumor samples, showed a significant correlation with nodal involvement in LCa patients (p < 0.01). CONCLUSION: This approach could allow us to set up a cost-effective and less invasive liquid biopsy protocol from a simple blood collection in order to early diagnose LCa and improve patients' outcomes and quality of life.

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.

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.

Protocol for synthesis of spherical silver nanoparticles with stable optical properties and characterization by transmission electron microscopy.

The synthesis of metallic plasmonic nanoparticles (NPs) faces challenges in stability and reproducibility, especially with silver. Here, we present a protocol for tunable synthesis of spherical silver NPs (AgNPs) with stable optical properties. We describe steps for preparing solutions, morphological characterization of AgNPs by transmission electron microscopy, and testing stability. AgNPs exhibit enduring stability and compatibility with various pH values. Moreover, they can be functionalized for optical biosensing applications, offering versatility in nanomaterial 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.

An All-Green Photo-Electrochemical Biosensor Using Microalgae Immobilized on Eco-Designed Lignin-Based Screen-Printed Electrodes to Detect Sustainable Nanoherbicides.

Herein, a novel completely green biosensor was designed exploiting both the biological and instrumental components made of eco-friendly materials for the detection of herbicides encapsulated into biodegradable nanoparticles for a sustainable agriculture. Similar nanocarriers, indeed, can deliver herbicides to the correct location, reducing the amount of active chemicals deposited in the plant, impacting the agricultural and food industries less. However, handling measurements of nanoherbicides is crucial to provide comprehensive information about their status in the agricultural fields to support farmers in decision-making. In detail, whole cells of the unicellular green photosynthetic alga Chlamydomonas reinhardtii UV180 mutant were immobilized by a green protocol on carbonized lignin screen-printed electrodes and integrated into a photo-electrochemical transductor for the detection of nanoformulated atrazine. Specifically, atrazine encapsulated into zein and chitosan doped poly-ε-caprolactone nanoparticles (atrazine-zein and atrazine-PCL-Ch) were analyzed following the current signals at a fixed applied potential of 0.8 V, in a range between 0.1 and 5 µM, indicating a linear relationship in the measured dose-response curves and a detection limit of 0.9 and 1.1 nM, respectively. Interference studies resulted in no interference from 10 ppb bisphenol A, 1 ppb paraoxon, 100 ppb arsenic, 20 ppb copper, 5 ppb cadmium, and 10 ppb lead at safety limits. Finally, no matrix effect was observed on the biosensor response from wastewater samples and satisfactory recovery values of 106 ± 8% and 93 ± 7% were obtained for atrazine-zein and atrazine-PCL-Ch, respectively. A working stability of 10 h was achieved.

Recent Advances in Stimuli-Responsive Hydrogel-Based Wound Dressing.

Polymeric materials have found increasing use in biomedical applications in the last decades. Among them, hydrogels represent the chosen class of materials to use in this field, in particular as wound dressings. They are generally non-toxic, biocompatible, and biodegradable, and they can absorb large amounts of exudates. Moreover, hydrogels actively contribute to skin repair promoting fibroblast proliferation and keratinocyte migration, allowing oxygen to permeate, and protecting wounds from microbial invasion. As wound dressing, stimuli-responsive systems are particularly advantageous since they can be active only in response to specific environmental stimuli (such as pH, light, ROS concentration, temperature, and glucose level). In this review, we briefly resume the human skin's structure and functions, as well as the wound healing phases; then, we present recent advances in stimuli-responsive hydrogels-based wound dressings. Lastly, we provide a bibliometric analysis of knowledge produced in the field.

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.

Microfluidic-Assisted Production of Gastro-Resistant Active-Targeted Diatomite Nanoparticles for the Local Release of Galunisertib in Metastatic Colorectal Cancer Cells.

The oral route is highly desirable for colorectal cancer (CRC) treatment because it allows concentrating the drug in the colon and achieving a localized effect. However, orally administered drugs are often metabolized in the liver, resulting in reduced efficacy and the need for higher doses. Nanoparticle-based drug delivery systems can be engineered to prevent the diffusion of the drug in the stomach, addressing the release at the target site, and enhancing the efficacy of the delivered drug. Here, an orally administrable galunisertib delivery system is developed with gelatin-covered diatomite nanoparticles targeting the ligand 1-cell adhesion molecule (L1-CAM) on metastatic cells, and further encapsulated in an enteric matrix by microfluidics. The gastro-resistant polymer protects the nanoparticles from the action of the digestive enzymes and allows for a sustained release of galunisertib at the intestinal pH. The efficacy of antibody-antigen interactions to drive the internalization of nanoparticles in the targeted cells is investigated in CRC cells expressing abnormal (SW620) or basal levels (Caco-2, HT29-MTX) of L1-CAM. The combination of local drug release and active targeting enhances the effect of the delivered galunisertib, which inhibits the migration of the SW620 cells with greater efficiency compared to the free drug.

Effect of microneedles shape on skin penetration and transdermal drug administration.

Microneedle (MN) patches are highly efficient and versatile tools for transdermal drug administration, in particular for pain-free, self-medication and rapid local applications. Diffraction ultraviolet (UV) light lithography offers an advanced method in fabricating poly(ethylene glycol)-based MNs with different shapes, by changing both the UV-light exposure time and photomask design. The exposure time interval is limited at obtaining conical structures with aspect ratio < 1:3, otherwise MNs exhibit reduced fracture load and poor indentation ability, not suitable for practical application. Therefore, this work is focused on a systematic analysis of the MN's base shapes effects on the structural characteristics, skin penetration and drug delivery. Analyzing four different base shapes (circle, triangle, square and star), it has been found that the number of vertices in the polygon base heavily affects these properties. The star-like MNs reveal the most efficient skin penetration ability (equal to 40 % of -their length), due to the edges action on the skin during the perforation. Furthermore, the quantification of the drug delivered by the MNs through ex-vivo porcine skin shows that the amounts of small molecules released over 24 h by star-like MNs coated by local anesthetic (Lidocaine) and an anti-inflammatory (Diclofenac epolamine) drugs are 1.5× and 2× higher than the circular-MNs, respectively.

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.

Antifungal and Antibiofilm Activity of Cyclic Temporin L Peptide Analogues against Albicans and Non-Albicans Candida Species.

Temporins are one of the largest families of antimicrobial peptides with both anti-inflammatory and antimicrobial activity. Herein, for a panel of cyclic temporin L isoform analogues, the antifungal and antibiofilm activities were determined against representative Candida strains, including C. albicans, C. glabrata, C. auris, C. parapsilosis and C. tropicalis. The outcomes indicated a significant anti-candida activity against planktonic and biofilm growth for four peptides (3, 7, 15 and 16). The absence of toxicity up to high concentrations and survival after infection were assessed in vivo by using Galleria mellonella larvae, and the correlation between conformation and cytotoxicity was investigated by fluorescence assays and circular dichroism (CD). By combining fluorescence spectroscopy, CD, dynamic light scattering, confocal and atomic force microscopy, the mode of action of four analogues was hypothesized. The results pinpointed that peptide 3 emerged as a non-toxic compound showing a potent antibiofilm activity and represents a promising compound for biomedical applications.

ROS-Generating Hyaluronic Acid-Modified Zirconium Dioxide-Acetylacetonate Nanoparticles as a Theranostic Platform for the Treatment of Osteosarcoma.

Materials that are able to produce free radicals have gained increasing attention for environmental and biomedical purposes. Free radicals, such as the superoxide anion (O2•-), act as secondary messengers in many physiological pathways, such as cell survival. Therefore, the production of free radicals over physiological levels has been exploited in the treatment of different types of cancer, including osteosarcoma (OS). In most cases, the production of reactive oxygen species (ROS) by materials is light-induced and requires the use of chemical photosensitisers, making it difficult and expensive. Here, for the first time, we propose photoluminescent hybrid ZrO2-acetylacetonate nanoparticles (ZrO2-acac NPs) that are capable of generating O2•- without light activation as an adjuvant for the treatment of OS. To increase the uptake and ROS generation in cancer cells, we modify the surface of ZrO2-acac NPs with hyaluronic acid (HA), which recognizes and binds to the surface antigen CD44 overexpressed on OS cells. Since these nanoparticles emit in the visible range, their uptake into cancer cells can be followed by a label-free approach. Overall, we show that the generation of O2•- is toxic to OS cells and can be used as an adjuvant treatment to increase the efficacy of conventional drugs.

Underwater Light Manipulation by the Benthic Diatom Ctenophora pulchella: From PAR Efficient Collection to UVR Screening.

Several species of diatoms, unicellular microalgae which constitute the main component of phytoplankton, are characterized by an impressive photosynthetic efficiency while presenting a noticeable tolerance versus exposure to detrimental UV radiation (UVR). In particular, the growth rate of the araphid diatom Ctenophora pulchella is not significantly affected by harsh treatments with UVR, even in absence of detectable, specific UV-absorbing pigments and even if it is not able to avoid high UV exposure by motility. In this work we applied a multi-disciplinary approach involving numerical computation, photonics, and biological parameters in order to investigate the possible role of the frustule, micro- and nano-patterned silica shell which encloses the cell, in the ability of C. pulchella to efficiently collect photosynthetic active radiation (PAR) and to simultaneously screen the protoplasm from UVR. The characterization of the photonic properties of the frustule has been accompanied by in vivo experiments conducted in water in order to investigate its function as optical coupler between light and plastids.

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.

Bioconjugation of Peptides to Hybrid Gold Nanoparticles.

Gold nanoparticles (AuNPs) can be produced by well-assessed synthesis methods and can show a high surface area-to-volume ratio, chemical inertness, high electron density, strong optical absorption as well as low toxicity. AuNPs have been conjugated with many different biomolecules for a wide range of biomedical applications. These applications require an increasingly complex level of surface decoration in order to achieve stability, efficacy, and specific functionalities. This chapter provides detailed instructions about the synthesis of AuNPs and bioconjugation strategies in order to obtain stable hybrid nanomaterials. The described biofunctionalization procedures are based on carbodiimide chemistry and ligand-exchange methods allowing the conjugation of Lys-peptide or Cys-peptide, respectively, to the AuNPs surface.

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.