A chiral trimethyl lock based on the vicinal disubstituent effect: prolonged release of camptothecin into cancer cells.

Synthesis and in vitro testing of a prodrug designed for the controlled delivery of the anticancer drug camptothecin within pancreatic cancer cells are reported. Our study reveals a non-conventional pharmacokinetic release characterized by an exponential pattern before reaching the half-life (t1/2) and a linear pattern thereafter. The release mechanism was triggered either by hydrolytic enzymes and/or by the acid microenvironment of cancer cells.

Cellulose Nanocrystals Show Anti-Adherent and Anti-Biofilm Properties against Oral Microorganisms.

Cellulose nanocrystals (CNCs) are cellulose-derived nanomaterials that can be easily obtained, e.g., from vegetable waste produced by circular economies. They show promising antimicrobial activity and an absence of side effects and toxicity. This study investigated the ability of CNCs to reduce microbial adherence and biofilm formation using in vitro microbiological models reproducing the oral environment. Microbial adherence by microbial strains of oral interest, Streptococcus mutans and Candida albicans, was evaluated on the surfaces of salivary pellicle-coated enamel disks in the presence of different aqueous solutions of CNCs. The anti-biofilm activity of the same CNC solutions was tested against S. mutans and an oral microcosm model based on mixed plaque inoculum using a continuous-flow bioreactor. Results showed the excellent anti-adherent activity of the CNCs against the tested strains from the lowest concentration tested (0.032 wt. %, p < 0.001). Such activity was significantly higher against S. mutans than against C. albicans (p < 0.01), suggesting a selective anti-adherent activity against pathogenic strains. At the same time, there was a minimal, albeit significant, anti-biofilm activity (0.5 and 4 wt. % CNC solution for S. mutans and oral microcosm, respectively, p = 0.01). This makes CNCs particularly interesting as anticaries agents, encouraging their use in the oral field.

Red-Emitting Carbon Quantum Dots for Biomedical Applications: Synthesis and Purification Issues of the Hydrothermal Approach.

The possibility of performing the synthesis of red-emitting carbon quantum dots (r-CDs), in a well-controllable, large scale and environmentally sustainable way is undoubtedly of fundamental importance, as it will pave the way to their employment in advanced medical large-scale applications. Knowledge of the difficulties involved in producing r-CDs with reproducible optical, structural, and chemical characteristics, might help in their large-scale production, making the process standardizable. In this work, we present an experimental study, also supported by results reported in the literature, on the issues encountered during the synthesis and post-synthesis purification treatments of r-CDS. We focused on the hydrothermal approach as it was found to be more suitable for future large-scale industrial applications. We propose three synthetic strategies and observed that employing p-phenylenediamine (p-PDA), as a precursor, the synthetic process showed low efficiency with low yields of r-CDs, large amounts of unreacted precursor, and reaction intermediates. Changing reaction parameters does not improve performance. The r-CDs obtained using citric acid (CA) and urea, as precursors, resulted to be sensitive to pH and difficult to separate from the reaction mixture. Furthermore, the proposed synthetic strategies show that the hydrothermal preparation of r-CDS requires approaches that are not fully sustainable.

Nitrogen-Doped Carbon Quantum Dots for Biosensing Applications: The Effect of the Thermal Treatments on Electrochemical and Optical Properties.

The knowledge of the ways in which post-synthesis treatments may influence the properties of carbon quantum dots (CDs) is of paramount importance for their employment in biosensors. It enables the definition of the mechanism of sensing, which is essential for the application of the suited design strategy of the device. In the present work, we studied the ways in which post-synthesis thermal treatments influence the optical and electrochemical properties of Nitrogen-doped CDs (N-CDs). Blue-emitting, N-CDs for application in biosensors were synthesized through the hydrothermal route, starting from citric acid and urea as bio-synthesizable and low-cost precursors. The CDs samples were thermally post-treated and then characterized through a combination of spectroscopic, structural, and electrochemical techniques. We observed that the post-synthesis thermal treatments show an oxidative effect on CDs graphitic N-atoms. They cause their partially oxidation with the formation of mixed valence state systems, [CDs]0+, which could be further oxidized into the graphitic N-oxide forms. We also observed that thermal treatments cause the decomposition of the CDs external ammonium ions into ammonia and protons, which protonate their pyridinic N-atoms. Photoluminescence (PL) emission is quenched.

Targeting GRP receptor: Design, synthesis and preliminary biological characterization of new non-peptide antagonists of bombesin.

We report the rational design, synthesis, and in vitro preliminary evaluation of a new small library of non-peptide ligands of Gastrin Releasing Peptide Receptor (GRP-R), able to antagonize its natural ligand bombesin (BN) in the nanomolar range of concentration. GRP-R is a transmembrane G-protein coupled receptor promoting the stimulation of cancer cell proliferation. Being overexpressed on the surface of different human cancer cell lines, GRP-R is ideal for the selective delivery to tumor cells of both anticancer drug and diagnostic devices. What makes very challenging the design of non-peptide BN analogues is that the 3D structure of the GRP-R is not available, which is the case for many membrane-bound receptors. Thus, the design of GRP-R ligands has to be based on the structure of its natural ligands, BN and GRP. We recently mapped the BN binding epitope by NMR and here we exploited the same spectroscopy, combined with MD, to define BN conformation in proximity of biological membranes, where the interaction with GRP-R takes place. The gained structural information was used to identify a rigid C-galactosidic scaffold able to support pharmacophore groups mimicking the BN key residues' side chains in a suitable manner for binding to GRP-R. Our BN antagonists represent hit compounds for the rational design and synthesis of new ligands and modulators of GRP-R. The further optimization of the pharmacophore groups will allow to increase the biological activity. Due to their favorable chemical properties and stability, they could be employed for the active receptor-mediated targeting of GRP-R positive tumors.

Pyranoid Spirosugars as Enzyme Inhibitors.

BACKGROUND: Pyranoid spirofused sugar derivatives represent a class of compounds with a significant impact in the literature. From the structural point of view, the rigidity inferred by the spirofused entity has made these compounds object of interest mainly as enzymatic inhibitors, in particular, carbohydrate processing enzymes. Among them glycogen phosphorylase and sodium glucose co-transporter 2 are important target enzymes for diverse pathological states. Most of the developed compounds present the spirofused entity at the C1 position of the sugar moiety; nevertheless, spirofused entities can also be found at other sugar ring positions. The main spirofused entities encountered are spiroacetals/thioacetals, spiro-hydantoin and derivatives, spiro-isoxazolines, spiro-aminals, spiro-lactams, spiro-oxathiazole and spiro-oxazinanone, but also others are present. OBJECTIVES: The present review focuses on the most explored synthetic strategies for the preparation of this class of compounds, classified according to the position and structure of the spirofused moiety on the pyranoid scaffold. Moreover, the structures are correlated to their main biological activities or to their role as chiral auxiliaries. CONCLUSION: It is clear from the review that, among the different derivatives, the spirofused structures at position C1 of the pyranoid scaffold are the most represented and possess the most relevant enzymatic inhibitor activities. Nevertheless, great efforts have been devoted to the introduction of the spirofused entity also in the other positions, mainly for the preparation of biologically active compounds but also for the synthesis of chiral auxiliaries useful in asymmetric reactions; examples of such auxiliaries are the spirofused chiral 1,3-oxazolidin-2-ones and 1,3-oxazolidine-2-thiones.

On-cell saturation transfer difference NMR study of Bombesin binding to GRP receptor.

We report the NMR characterization of the molecular interaction between Gastrin Releasing Peptide Receptor (GRP-R) and its natural ligand bombesin (BN). GRP-R is a transmembrane G-protein coupled receptor promoting the stimulation of cancer cell proliferation; in addition, being overexpressed on the surface of different human cancer cell lines, it is ideal for the development of new strategies for the selective targeted delivery of anticancer drugs and diagnostic devices to tumor cells. However, the design of new GRP-R binders requires structural information on receptor interaction with its natural ligands. The experimental protocol presented herein, based on on-cell STD NMR techniques, is a powerful tool for the screening and the epitope mapping of GRP-R ligands aimed at the development of new anticancer and diagnostic tools. Notably, the study can be carried out in a physiological environment, at the surface of tumoral cells overespressing GRP-R. Moreover, to the best of our knowledge, this is the first example of an NMR experiment able to detect and investigate the structural determinants of BN/GRP-R interaction.

PLGA Based Nanoparticles for the Monocyte-Mediated Anti-Tumor Drug Delivery System.

Together with the development of new therapeutic agents, innovation in the delivery system of anti-tumor drugs is required to increase tumor-specificity and avoid unexpected toxicity. To achieve higher efficiency, we combined a live cell-mediated drug delivery system with nanotechnology, with the aim to prove that blood monocytes can be a cargo to deliver antitumor drugs encapsulated in Polymeric poly(D, L-lactide-co-glycolide) acid based nanoparticles (PLGA NPs). In this study, we have characterized how isolated purified monocytes efficiently internalize PLGA-NPs and have imaged in vivo their trafficking upon intravenous injection in tumor-bearing mice. Monocytes carrying PLGA-Cy7 NPs were able to reach the tumor site, with superior efficiency than free PLGA-Cy7 NPs, and the bio-distribution analysis confirmed that tumors were the most reached among peripheral tissues. We further demonstrate that monocytes carrying Doxorubicin encapsulated PLGA NPs (PLGA-Doxo) induced strong killing of co-cultured tumor cells. Our studies provide proof-of-concept evidence that monocytes can be exploited in approaches of live cell-mediated drug delivery systems for tumor therapy.

Hexosamine pathway inhibition overcomes pancreatic cancer resistance to gemcitabine through unfolded protein response and EGFR-Akt pathway modulation.

Different evidence has indicated metabolic rewiring as a necessity for pancreatic cancer (PC) growth, invasion, and chemotherapy resistance. A relevant role has been assigned to glucose metabolism. In particular, an enhanced flux through the Hexosamine Biosynthetic Pathway (HBP) has been tightly linked to PC development. Here, we show that enhancement of the HBP, through the upregulation of the enzyme Phosphoacetylglucosamine Mutase 3 (PGM3), is associated with the onset of gemcitabine (GEM) resistance in PC. Indeed, mRNA profiles of GEM sensitive and resistant patient-derived tumor xenografts (PDXs) indicate that PGM3 expression is specifically increased in GEM-resistant PDXs. Of note, PGM3 results also overexpressed in human PC tissues as compared to paired adjacent normal tissues and its higher expression in PC patients is associated with worse median overall survival (OS). Strikingly, genetic or pharmacological PGM3 inhibition reduces PC cell growth, migration, invasion, in vivo tumor growth and enhances GEM sensitivity. Thus, combined treatment between a specific inhibitor of PGM3, named FR054, and GEM results in a potent reduction of xenograft tumor growth without any obvious side effects in normal tissues. Mechanistically, PGM3 inhibition, reducing protein glycosylation, causes a sustained Unfolded Protein Response (UPR), a significant attenuation of the pro-tumorigenic Epidermal Growth Factor Receptor (EGFR)-Akt axis, and finally cell death. In conclusion this study identifies the HBP as a metabolic pathway involved in GEM resistance and provides a strong rationale for a PC therapy addressing the combined treatment with the PGM3 inhibitor and GEM.

Glycan Carriers As Glycotools for Medicinal Chemistry Applications.

Carbohydrates are one of the most powerful and versatile classes of biomolecules that nature uses to regulate organisms' biochemistry, modulating plenty of signaling events within cells, triggering a plethora of physiological and pathological cellular behaviors. In this framework, glycan carrier systems or carbohydrate-decorated materials constitute interesting and relevant tools for medicinal chemistry applications. In the last few decades, efforts have been focused, among others, on the development of multivalent glycoconjugates, biosensors, glycoarrays, carbohydrate-decorated biomaterials for regenerative medicine, and glyconanoparticles. This review aims to provide the reader with a general overview of the different carbohydrate carrier systems that have been developed as tools in different medicinal chemistry approaches relying on carbohydrate-protein interactions. Given the extent of this topic, the present review will focus on selected examples that highlight the advancements and potentialities offered by this specific area of research, rather than being an exhaustive literature survey of any specific glyco-functionalized system.

bioNMR-based identification of natural anti-Aß compounds in Peucedanum ostruthium.

The growing interest in medicinal plants for the identification of new bioactive compounds and the formulation of new nutraceuticals and drugs prompted us to develop a powerful experimental approach allowing the detailed metabolic profiling of complex plant extracts, the identification of ligands of macromolecular targets of biomedical relevance and a preliminary characterization of their biological activity. To this end, we selected Peucedanum ostruthium, a plant traditionally employed in Austria and Italy for its several potential therapeutic applications, as case study. We combined the use of NMR and UPLC-HR-MS for the identification of the metabolites present in its leaves and rhizome extracts. Due to the significant content of polyphenols, particularly chlorogenic acids, recently identified as anti-amyloidogenic compounds, polyphenols-enriched fractions were prepared and tested for their ability to prevent Aß1-42 peptide aggregation and neurotoxicity in a neuronal human cell line. STD-NMR experiments allowed the detailed identification of Aß oligomers' ligands responsible for the anti-amyloidogenic activity. These data provide experimental protocols and structural information suitable for the development of innovative molecular tools for prevention, therapy and diagnosis of Alzheimer's disease.

Glycofunctionalization of Poly(lactic- co-glycolic acid) Polymers: Building Blocks for the Generation of Defined Sugar-Coated Nanoparticles.

A set of poly(lactic- co-glycolic acid) polymers functionalized with different monosaccharides as well as glycodendrimers and surface-decorated nanoparticles (NPs) were synthesized and characterized. The functionalization of the polymer was carried out through amide bond formation with amino-modified sugar monomers and through a biocompatible chemoselective method exploiting the reducing end of a free sugar. The assemblage of the NPs adopting a nanoprecipitation method was straightforward and allowed the preparation of sugars/sugar dendrimer coated NPs.

Inhibition of the Hexosamine Biosynthetic Pathway by targeting PGM3 causes breast cancer growth arrest and apoptosis.

Cancer aberrant N- and O-linked protein glycosylation, frequently resulting from an augmented flux through the Hexosamine Biosynthetic Pathway (HBP), play different roles in tumor progression. However, the low specificity and toxicity of the existing HBP inhibitors prevented their use for cancer treatment. Here we report the preclinical evaluation of FR054, a novel inhibitor of the HBP enzyme PGM3, with a remarkable anti-breast cancer effect. In fact, FR054 induces in different breast cancer cells a dramatic decrease in cell proliferation and survival. In particular, in a model of Triple Negative Breast Cancer (TNBC) cells, MDA-MB-231, we show that these effects are correlated to FR054-dependent reduction of both N- and O-glycosylation level that cause also a strong reduction of cancer cell adhesion and migration. Moreover we show that impaired survival of cancer cells upon FR054 treatment is associated with the activation of the Unfolded Protein Response (UPR) and accumulation of intracellular ROS. Finally, we show that FR054 suppresses cancer growth in MDA-MB-231 xenograft mice, supporting the advantage of targeting HBP for therapeutic purpose and encouraging further investigation about the use of this small molecule as a promising compound for breast cancer therapy.

Flavonoids in the Treatment of Alzheimer's and Other Neurodegenerative Diseases.

Flavonoids are phytochemicals present in almost all terrestrial plants and, as a consequence, in plant-based foods, and thus consumed by humans through diet. Recent evidences suggest that several flavonoids have positive effects against dementia and Alzheimer's disease, reversing age-related declines in neurocognitive performances. In this review, we provide a general classification of natural and synthetic flavonoids, a description of their physico-chemical properties, in particular their redox properties and stability, and an extensive overview about their biological activities and structure-activity relationship in the field of neurodegenerative diseases. In addition, a section will be dedicated to the synthetic strategies for the preparation of bioactive derivatives. This information will be essential for the design and development of new drugs that can improve brain functions.

A Randomized, Double-Blind, Placebo-Controlled Trial: The Efficacy of Multispecies Probiotic Supplementation in Alleviating Symptoms of Irritable Bowel Syndrome Associated with Constipation.

Background and Aim. The efficacy of supplementation treatment with two multispecies probiotic formulates on subjects diagnosed with IBS-C and the assessment of their gut microbiota were investigated. Methods. A randomized, double-blind, three-arm parallel group trial was carried out on 150 IBS-C subjects divided into three groups (F_1, F_2, and F_3). Each group received a daily oral administration of probiotic mixtures (for 60 days) F_1 or F_2 or placebo F_3, respectively. Fecal microbiological analyses were performed by species-specific qPCR to assess the different amount of probiotics. Results. The percentage of responders for each symptom was higher in the probiotic groups when compared to placebo group during the treatment period (t60) and was maintained quite similar during the follow-up period (t90). Fecal analysis demonstrated that probiotics of the formulations increased during the times of treatment only in fecal DNA from subjects treated with F_1 and F_2 and not with F_3, and the same level was maintained during the follow-up period. Conclusions. Multispecies probiotic supplementations are effective in IBS-C subjects and induce a different assessment in the composition of intestinal microbiota. This clinical study is registered with the clinical study registration number ISRCTN15032219.

(18)F-labeling syntheses and preclinical evaluation of functionalized nanoliposomes for Alzheimer's disease.

The aim of the present study was to synthesize functionalized (18)F-labeled NLs ((18)F-NLs) and evaluate their biological behavior in mouse models of Alzheimer's disease (AD) using positron emission tomography (PET) and ex vivo brain autoradiography. (18)F-fluorine was introduced to (18)F-NLs either by using a core forming (18)F-lipid or by encapsulating a (18)F-tracer, (18)F-treg-curcumin inside the NLs. Phosphatidic acid (PA) and curcumin derivative (Curc) functionalized (18)F-NLs with or without additional mApoE functionalization were produced using thin film hydration. The biodistribution and ß-amyloid plaque-binding ability of (18)F-NLs were studied in wild type mice and AD mouse models using in vivo PET imaging and ex vivo brain autoradiography at 60min after (18)F-NL injection. Functionalized (18)F-NLs were successfully synthesized. The preclinical evaluation in mice showed that the functional group affected the biodistribution of (18)F-NLs. Further functionalization with mApoE increased the brain-to-blood ratio of (18)F-NLs but the overall brain uptake remained low with all functionalized (18)F-NLs. The liposomal encapsulation of (18)F-treg-curcumin was not successful and preclinical results of encapsulated (18)F-treg-curcumin and plain (18)F-treg-curcumin were identical. Although the studied functionalized (18)F-NLs were not suitable for PET imaging as such, the synthesis techniques introduced in this study can be utilized to modify the biological behavior of (18)F-labeled NLs.

Structural Modifications of cis-Glycofused Benzopyran Compounds and Their Influence on the Binding to Amyloid-ß Peptide.

A small library of glycofused tricyclic compounds with a central pyran ring chemically modified in the position para to the ring oxygen has been synthesised. The influence of the chemical modification on the structural conformation of the compounds and on their ability to bind Aß peptide has been evaluated respectively using molecular mechanics (MM) and molecular dynamics (MD) simulations, and STD NMR spectroscopy. The introduction of particularly polar/charged groups leads to the loss of binding ability, without a significant change in the conformation, whilst other substitutions does not significantly affect either the structural conformation or the binding.

Organ Distribution and Bone Tropism of Cellulose Nanocrystals in Living Mice.

Their physicochemical properties and relatively low cost make cellulose nanocrystals (CNCs) a potential candidate for future large-scale production in many fields including nanomedicine. Prior to a sustained and responsible development as theranostic agents, robust and reliable data concerning their safety, biocompatibility, and tissue distribution should be provided. In the present study, CNCs were extracted from Whatman filters functionalized with a fluorescent dye, and their interaction with living organisms has been thoroughly assessed. Our experimental evidence demonstrated that CNCs (1) are well tolerated by healthy mice after systemic injection; (2) are rapidly excreted, thus avoiding bioaccumulation in filter organs such as the kidneys and liver; (3) transiently migrate in bones; and (4) are able to penetrate in the cytoplasm of cancer cells without inducing material-related detrimental effects in terms of cell survival. Our results strongly suggest that the peculiar tropism to the bones is due to the chemical interaction between the Ca(2+) of the bone matrix and the active surface of negatively-charged CNCs. This feature, together with the ability to penetrate cancer cells, makes CNCs a potential nanodevice for theranostics in bone tumors.

Comparison of Various Types of Ligand Decorated Nanoliposomes for their Ability to Inhibit Amyloid Aggregation and to Reverse Amyloid Cytotoxicity.

Three different amyloid targeting ligands, previously shown to exhibit amyloid specific properties, have been used to develop amyloid -targeted nanoliposomes (AT-NLs. For this a MAb against Aß-peptides (Aß-MAb (immobilized on NLs at 0.015 and 0.05 mol %, and two different curcumin-lipid derivatives were attached to the surface of preformed NLs or incorporated in NL membranes during their formation. Following physicochemical characterization, these AT-NLs were studied for their ability to inhibit or delay amyloid peptide aggregation -using the thioflavin-T assay, and for their potential to reverse amyloid-induced (and Zn, or, amyloid + Zn cytotoxicity, on wild type (N2aWT and transformed (N2aAPP neuroblastoma cells, applying the MTT assay. Experimental results reveal that all formulations were found to strongly delay amyloid peptide aggregation (with no significant differences between the different AT-NL types. However, although Aß-MAb-NLs significantly reversed amyloid-induced cytotoxicity in all cases, both curcumin-NL types did not reverse Zn-induced, nor Zn+Aß-induced cytotoxicity in N2aWT cells, suggesting lower activity against synthetic-Aß peptides (compared to endogenous Aß peptides; perhaps due to different affinity towards different (aggregation stages of peptide species (monomers, oligomers, fibrils, etc. Taken into account that the aggregation stage of amyloid species is an important determinant of their toxicity, the importance of the affinity of each AT-NL type towards specific species, is highlighted.

Nanoliposomes presenting on surface a cis-glycofused benzopyran compound display binding affinity and aggregation inhibition ability towards Amyloid ß1-42 peptide.

Nanoliposomes decorated on their surface with ligands for Aß-peptides, the key morphological features of Alzheimer's disease (AD), have been synthesized and characterized for their ability to target Aß-peptide aggregates. A tricyclic benzopyrane-glycofused structure has been exploited as Aß-peptide ligand, which was linked to liposomes via a copper-free, chemoselective, biocompatible click chemistry reaction. The tricyclic-decorated liposomes presented a mean diameter in the nanomolar range (150-200 nm), a negative z-potential and a good stability, at least up to one month. Integrity studies performed in the presence of serum proteins indicated that these decorated nanoliposomes fulfill the requirements for in vivo applications. NMR experiments carried out with Aß1-42 oligomers using both surface functionalized and plain (control) liposomes, revealed that the binding ability of the nanoliposomes was mediated by the presence of the tricyclic ligand on their surface. Finally ThT assay carried out with tricyclic-decorated liposomes showed significant decrease in thioflavine T fluorescence after 24 h, suggesting a significant inhibition/delay of Aß1-42 aggregation.