Thermosensitive In Situ Gelling Poloxamers/Hyaluronic Acid Gels for Hydrocortisone Ocular Delivery.

This study endeavored to overcome the physiological barriers hindering optimal bioavailability in ophthalmic therapeutics by devising drug delivery platforms that allow therapeutically effective drug concentrations in ocular tissues for prolonged times. Thermosensitive drug delivery platforms were formulated by blending poloxamers (F68 and F127) with low-molecular-weight hyaluronic acid (HA) in various concentrations and loaded with hydrocortisone (HC). Among the formulations examined, only three were deemed suitable based on their desirable gelling properties at a temperature close to the eye's surface conditions while also ensuring minimal gelation time for swift ocular application. Rheological analyses unveiled the ability of the formulations to develop gels at suitable temperatures, elucidating the gel-like characteristics around the physiological temperature essential for sustained drug release. The differential scanning calorimetry findings elucidated intricate hydrogel-water interactions, indicating that HA affects the water-polymer interactions within the gel by increasing the platform hydrophilicity. Also, in vitro drug release studies demonstrated significant hydrocortisone release within 8 h, governed by an anomalous transport mechanism, prompting further investigation for optimized release kinetics. The produced platforms offer promising prospects for efficacious ocular drug delivery, addressing pivotal challenges in ocular therapeutics and heralding future advancements in the domain.

Unravelling the role of lipid composition on liposome-protein interactions.

Upon in vivo administration of nanoparticles, a protein corona forms on their surface and affects their half-life in circulation, biodistribution properties, and stability; in turn, the composition of the protein corona depends on the physico-chemical properties of the nanoparticles. We have previously observed lipid composition-dependent in vitro and in vivo microRNA delivery from lipid nanoparticles. Here, we carried out an extensive physico-chemical characterisation to understand the role of the lipid composition on the in vivo fate of lipid-based nanoparticles. We used a combination of differential scanning calorimetry (DSC), membrane deformability measurements, isothermal titration calorimetry (ITC), and dynamic light scattering (DLS) to probe the interactions between the nanoparticle surface and bovine serum albumin (BSA) as a model protein. The lipid composition influenced membrane deformability, improved lipid intermixing, and affected the formation of lipid domains while BSA binding to the liposome surface was affected by the PEGylated lipid content and the presence of cholesterol. These findings highlight the importance of the lipid composition on the protein-liposome interaction and provide important insights for the design of lipid-based nanoparticles for drug delivery applications.

Bacterial model membranes under the harsh subsurface conditions of Mars.

Biomembranes are a key component of all living systems. Most research on membranes is restricted to ambient physiological conditions. However, the influence of extreme conditions, such as the deep subsurface on Earth or extraterrestrial environments, is less well understood. The deep subsurface of Mars is thought to harbour high concentrations of chaotropic salts in brines, yet we know little about how these conditions would influence the habitability of such environments. Here, we investigated the combined effects of high concentrations of Mars-relevant salts, including sodium and magnesium perchlorate and sulphate, and high hydrostatic pressure on the stability, structure, and function of a bacterial model membrane. To this end, several biophysical techniques have been employed, including calorimetry, fluorescence and CD spectroscopy, confocal microscopy, and small-angle X-ray scattering. We demonstrate that sulphate and perchlorate salts affect the properties of the membrane differently, depending on the counterion present (Na+vs. Mg2+). We found that the perchlorates, which are believed to be abundant salts in the Martian environment, induce a more hydrated and less ordered membrane, strongly favouring the physiologically relevant fluid-like phase of the membrane even under high-pressure stress. Moreover, we show that the activity of the phospholipase A2 is strongly modulated by both high pressure and salt. Compellingly, in the presence of the chaotropic perchlorate, the enzymatic reaction proceeded at a reasonable rate even in the presence of condensing Mg2+ and at high pressure, suggesting that bacterial membranes could still persist when challenged to function in such a highly stressed Martian environment.

Exploring the G-quadruplex binding and unwinding activity of the bacterial FeS helicase DinG.

Despite numerous reports on the interactions of G-quadruplexes (G4s) with helicases, systematic analysis addressing the selectivity and specificity of each helicase towards a variety of G4 topologies are scarce. Among the helicases able to unwind G4s are those containing an iron-sulphur (FeS) cluster, including both the bacterial DinG (found in E. coli and several pathogenic bacteria) and the medically important eukaryotic homologues (XPD, FancJ, DDX11 and RTEL1). We carried out a detailed study of the interactions between the E. coli DinG and a variety of G4s, by employing physicochemical and biochemical methodologies. A series of G4-rich sequences from different genomic locations (promoter and telomeric regions), able to form unimolecular G4 structures with diverse topologies, were analyzed (c-KIT1, KRAS, c-MYC, BCL2, Tel23, T30695, Zic1). DinG binds to most of the investigated G4s with little discrimination, while it exhibits a clear degree of unwinding specificity towards different G4 topologies. Whereas previous reports suggested that DinG was active only on bimolecular G4s, here we show that it is also able to bind to and resolve the more physiologically relevant unimolecular G4s. In addition, when the G4 structures were stabilized by ligands (Pyridostatin, PhenDC3, BRACO-19 or Netropsin), the DinG unwinding activity decreased and in most cases was abolished, with a pattern that is not simply explained by a change in binding affinity. Overall, these results have important implications for the biochemistry of helicases, strongly suggesting that when analysing the G4 unwinding property of an enzyme, it is necessary to investigate a variety of G4 substrates.

Truncated Analogues of a G-Quadruplex-Forming Aptamer Targeting Mutant Huntingtin: Shorter Is Better!

Two analogues of the MS3 aptamer, which was previously shown to have an exquisite capability to selectively bind and modulate the activity of mutant huntingtin (mHTT), have been here designed and evaluated in their physicochemical and biological properties. Featured by a distinctive propensity to form complex G-quadruplex structures, including large multimeric aggregates, the original 36-mer MS3 has been truncated to give a 33-mer (here named MS3-33) and a 17-mer (here named MS3-17). A combined use of different techniques (UV, CD, DSC, gel electrophoresis) allowed a detailed physicochemical characterization of these novel G-quadruplex-forming aptamers, tested in vitro on SH-SY5Y cells and in vivo on a Drosophila Huntington's disease model, in which these shorter MS3-derived oligonucleotides proved to have improved bioactivity in comparison with the parent aptamer.

Fighting the Huntington's Disease with a G-Quadruplex-Forming Aptamer Specifically Binding to Mutant Huntingtin Protein: Biophysical Characterization, In Vitro and In Vivo Studies.

A set of guanine-rich aptamers able to preferentially recognize full-length huntingtin with an expanded polyglutamine tract has been recently identified, showing high efficacy in modulating the functions of the mutated protein in a variety of cell experiments. We here report a detailed biophysical characterization of the best aptamer in the series, named MS3, proved to adopt a stable, parallel G-quadruplex structure and show high nuclease resistance in serum. Confocal microscopy experiments on HeLa and SH-SY5Y cells, as models of non-neuronal and neuronal cells, respectively, showed a rapid, dose-dependent uptake of fluorescein-labelled MS3, demonstrating its effective internalization, even in the absence of transfecting agents, with no general cytotoxicity. Then, using a well-established Drosophila melanogaster model for Huntington's disease, which expresses the mutated form of human huntingtin, a significant improvement in the motor neuronal function in flies fed with MS3 was observed, proving the in vivo efficacy of this aptamer.

Conformational plasticity of DNA secondary structures: probing the conversion between i-motif and hairpin species by circular dichroism and ultraviolet resonance Raman spectroscopies.

The promoter regions of important oncogenes such as BCL2 and KRAS contain GC-rich sequences that can form distinctive noncanonical DNA structures involved in the regulation of transcription: G-quadruplexes on the G-rich strand and i-motifs on the C-rich strand. Interestingly, BCL2 and KRAS promoter i-motifs are highly dynamic in nature and exist in a pH-dependent equilibrium with hairpin and even with hybrid i-motif/hairpin species. Herein, the effects of pH and presence of cell-mimicking molecular crowding conditions on conformational equilibria of the BCL2 and KRAS i-motif-forming sequences were investigated by ultraviolet resonance Raman (UVRR) and circular dichroism (CD) spectroscopies. Multivariate analysis of CD data was essential to model the presence and identity of the species involved. Analysis of UVRR spectra measured as a function of pH, performed also by the two-dimensional correlation spectroscopy (2D-COS) technique, showed the role of several functional groups in the DNA conformational transitions, and provided structural and dynamic information. Thus, the UVRR investigation of intramolecular interactions and of local and environmental dynamics in promoting the different species induced by the solution conditions provided valuable insights into i-motif conformational transitions. The combined use of the two spectroscopic tools is emphasized by the relevant possibility of working in the same DNA concentration range and by the heterospectral UVRR/CD 2D-COS analysis. The results of this study shed light on the factors that can influence at the molecular level the equilibrium between the different conformational species putatively involved in the oncogene expression.

Complexation of Cyclodextrins with Benzoic Acid in Water-Organic Solvents: A Solvation-Thermodynamic Approach.

The aim of this research is to obtain new data about the complexation between ß-cyclodextrin (ß-CD) and benzoic acid (BA) as a model reaction of the complex formation of hydrophobic molecules with cyclodextrins (CDs) in various media. This research may help developing cyclodextrin-based pharmaceutical formulations through the choice of the appropriate solvent mixture that may be employed in the industrial application aiming to control the reactions/processes in liquid phase. In this paper, NMR results for the molecular complex formation between BA and ß-CD ([BA⊂ß-CD]) in D2O-DMSO-d6 and in D2O-EtOH have shown that the stability of the complex in the H2O-DMSO-d6 varies within the experimental error, while decreases in H2O-EtOH. Changes in the Gibbs energy of BA resolvation in water and water-dimethylsulfoxide mixtures have been obtained and have been used in the analysis of the reagent solvation contributions into the Gibbs energy changes of the [BA⊂ß-CD] molecular complex formation. Quantum chemical calculations of the interaction energy between ß-CD and BA as well as the structure of the [BA⊂ß-CD] complex and the energy of ß-CD and BA interaction in vacuum and in the medium of water, methanol and dimethylsulfoxide solvents are carried out. The stability of [BA⊂ß-CD] complex in H2O-EtOH and H2O-DMSO solvents, obtained by different methods, are compared. The thermodynamic parameters of the [BA⊂ß-CD] molecular complexation as well as the reagent solvation contributions in H2O-EtOH and H2O-DMSO mixtures were analyzed by the solvation-thermodynamic approach.

On the thermodynamics of folding of an i-motif DNA in solution under favorable conditions.

Under slightly acidic conditions, cytosine-rich DNA sequences can form non-canonical secondary structures called i-motifs, which occur as four stretches of cytosine repeats form hemi-protonated C·C+ base pairs. The growing interest in the i-motif structures as important components in functional DNA-based nanotechnology or as potential targets of anticancer drugs, increases the need for a deep understanding of the energetics of their structural transitions. Here, a combination of spectroscopic and calorimetric techniques is used to unravel the thermodynamics of folding of an i-motif DNA under favorable conditions. The results give new insights into the energetic aspects of i-motifs and show that thermodynamic and thermal stability are related but not identical properties of such DNA structures.

Molecular Docking and Biophysical Studies for Antiproliferative Assessment of Synthetic Pyrazolo-Pyrimidinones Tethered with Hydrazide-Hydrazones.

Chemotherapy represents the most applied approach to cancer treatment. Owing to the frequent onset of chemoresistance and tumor relapses, there is an urgent need to discover novel and more effective anticancer drugs. In the search for therapeutic alternatives to treat the cancer disease, a series of hybrid pyrazolo[3,4-d]pyrimidin-4(5H)-ones tethered with hydrazide-hydrazones, 5a-h, was synthesized from condensation reaction of pyrazolopyrimidinone-hydrazide 4 with a series of arylaldehydes in ethanol, in acid catalysis. In vitro assessment of antiproliferative effects against MCF-7 breast cancer cells, unveiled that 5a, 5e, 5g, and 5h were the most effective compounds of the series and exerted their cytotoxic activity through apoptosis induction and G0/G1 phase cell-cycle arrest. To explore their mechanism at a molecular level, 5a, 5e, 5g, and 5h were evaluated for their binding interactions with two well-known anticancer targets, namely the epidermal growth factor receptor (EGFR) and the G-quadruplex DNA structures. Molecular docking simulations highlighted high binding affinity of 5a, 5e, 5g, and 5h towards EGFR. Circular dichroism (CD) experiments suggested 5a as a stabilizer agent of the G-quadruplex from the Kirsten ras (KRAS) oncogene promoter. In the light of these findings, we propose the pyrazolo-pyrimidinone scaffold bearing a hydrazide-hydrazone moiety as a lead skeleton for designing novel anticancer compounds.

KRAS Promoter G-Quadruplexes from Sequences of Different Length: A Physicochemical Study.

DNA G-quadruplexes (G4s) form in relevant genomic regions and intervene in several biological processes, including the modulation of oncogenes expression, and are potential anticancer drug targets. The human KRAS proto-oncogene promoter region contains guanine-rich sequences able to fold into G4 structures. Here, by using circular dichroism and differential scanning calorimetry as complementary physicochemical methodologies, we compared the thermodynamic stability of the G4s formed by a shorter and a longer version of the KRAS promoter sequence, namely 5'-AGGGCGGTGTGGGAATAGGGAA-3' (KRAS 22RT) and 5'-AGGGCGGTGTGGGAAGAGGGAAGAGGGGGAGG-3' (KRAS 32R). Our results show that the unfolding mechanism of KRAS 32R is more complex than that of KRAS 22RT. The different thermodynamic stability is discussed based on the recently determined NMR structures. The binding properties of TMPyP4 and BRACO-19, two well-known G4-targeting anticancer compounds, to the KRAS G4s were also investigated. The present physicochemical study aims to help in choosing the best G4 target for potential anticancer drugs.

Improved Anti-Prion Nucleic Acid Aptamers by Incorporation of Chemical Modifications.

Nucleic acid aptamers are innovative and promising candidates to block the hallmark event in the prion diseases, that is the conversion of prion protein (PrP) into an abnormal form; however, they need chemical modifications for effective therapeutic activity. This communication reports on the development and biophysical characterization of a small library of chemically modified G-quadruplex-forming aptamers targeting the cellular PrP and the evaluation of their anti-prion activity. The results show the possibility of enhancing anti-prion aptamer properties through straightforward modifications.

Optimization of a Low-Cost, Sensitive PNA Clamping PCR Method for JAK2 V617F Variant Detection.

BACKGROUND: The JAK2 V617F variant is diagnostic for myeloproliferative neoplasms, a group of clonal disorders of hematopoietic stem and progenitor cells. Although several approaches have been developed to detect the variant, a gold standard diagnostic method has not yet been defined. We describe a simple, fast, and cost-effective PCR-based approach that enhances test specificity and sensitivity by blocking the amplification of the large excess of wild-type DNA. METHODS: The method involves using an oligo peptide nucleic acid (PNA) perfectly matching its corresponding DNA sequence. The PCR protocol was optimized by collecting a detailed thermodynamic data set on PNA-DNA wild-type duplexes by circular dichroism melting experiments. The specificity and sensitivity of PNA clamping PCR were assessed by genotyping 50 patients with myeloproliferative neoplasm who carried the JAK2 V617F variant and 50 healthy donors. RESULTS: The optimized protocol enabled selective amplification of the variant alleles, achieving maximum sensitivity (100%) and specificity (100%). Analytical sensitivity was 0.05% of variant alleles as assessed by serial dilutions of DNA from the HEL cell line (which carries the JAK2 V617F variant) mixed to wild-type DNA from healthy donors. The JAK2 V617F variant test performed according to this method has better diagnostic performance than its 2 main PCR-based competitors, at much lower cost. CONCLUSIONS: High sensitivity and specificity and cost-effectiveness make PNA clamping PCR a useful testing platform for the detection of minor allele variants in small-scale diagnostic laboratories. It promises to improve patient care while enabling significant healthcare savings.

Targeting the KRAS oncogene: Synthesis, physicochemical and biological evaluation of novel G-Quadruplex DNA binders.

The oncogene KRAS is involved in the pathogenesis of many tumors such as pancreatic, lung and colorectal cancers, thereby representing a relevant target for the treatment of these diseases. The KRAS P1 promoter contains a nuclease hypersensitive, guanine-rich sequence able to fold into a G-quadruplex motif (G4). The stabilization of this G4 structure by small molecules is emerging as a feasible approach to downregulate KRAS expression. Here, a set of novel stabilizing molecules was identified through a virtual screening campaign on the NMR structure of the 22-mer KRAS G4. The most promising hits were then submitted to structure-activity relationships studies which allowed improving their binding affinity and selectivity over double helix DNA and different G4 topologies. The best derivative (19) underwent fluorescence titration experiments and further computational studies to disclose its binding mechanism to KRAS G4. Finally, biological assays showed that this compound is capable to reduce the viability of colorectal cancer cells in which mutated KRAS acts as a driver oncogene. Thus, 19 might represent the prototype of a new class of drugs for the treatment of tumors that, expressing mutated forms of KRAS, are refractory to current therapeutic regimens.

Ligand binding to G-quadruplex DNA: new insights from ultraviolet resonance Raman spectroscopy.

G-Quadruplexes (G4s) are noncanonical nucleic acid structures involved in the regulation of several biological processes of many organisms. The rational design of G4-targeting molecules developed as potential anticancer and antiviral therapeutics is a complex problem intrinsically due to the structural polymorphism of these peculiar DNA structures. The aim of the present work is to show how Ultraviolet Resonance Raman (UVRR) spectroscopy can complement other techniques in providing valuable information about ligand/G4 interactions in solution. Here, the binding of BRACO-19 and Pyridostatin - two of the most potent ligands - to selected biologically relevant G4s was investigated by polarized UVRR scattering at 266 nm. The results give new insights into the binding mode of these ligands to G4s having different sequences and topologies by performing an accurate analysis of peaks assigned to specific groups and their changes upon binding. Indeed, the UVRR data not only show that BRACO-19 and Pyridostatin interact with different G4 sites, but also shed light on the ligand and G4 chemical groups really involved in the interaction. In addition, UVRR results complemented by circular dichroism data clearly indicate that the binding mode of a ligand can also depend on the conformation(s) of the target G4. Overall, these findings demonstrate the utility of using UVRR spectroscopy in the investigation of G4s and G4-ligand interactions in solution.

Selective binding of a bioactive porphyrin-based photosensitizer to the G-quadruplex from the KRAS oncogene promoter.

BACKGROUND: The G-quadruplex-forming sequence within the KRAS proto-oncogene P1 promoter is a promising target for anticancer therapy. Porphyrin derivatives are among the most rewarding G-quadruplex binders. They can also behave as photosensitizers. METHODS: Three water-soluble, positively charged porphyrin-like compounds were synthesized and tested for their interaction with the KRAS G-quadruplex by circular dichroism, fluorescence, and molecular docking calculations. For a comparison of ligands binding affinity and selectivity, TMPyP4 was taken as a reference. RESULTS: One out of the three tested compounds proved biological activity and selectivity for G-quadruplex over duplex DNA. It also showed to discriminate between different G-quadruplex topologies, with a preference for the parallel over antiparallel conformation. Molecular docking studies suggested a preferential binding to the 3'-end of the KRAS G-quadruplex driven through π-π stacking interactions. Biological assays also revealed a good photodynamic-induced cytotoxicity on HeLa cells. CONCLUSIONS: The reported results show that these porphyrin-like compounds could actually give the basis for the development of G-quadruplex ligands with effective photodynamic-induced cytotoxicity on cancer cells. GENERAL SIGNIFICANCE: The possibility of obtaining photosensitizers with improved physico-chemical features and able to selectively target G-quadruplexes is a very interesting perspective to develop new therapeutic agents.

Crowding and conformation interplay on human DNA G-quadruplex by ultraviolet resonant Raman scattering.

The G-quadruplex-forming telomeric sequence (TTAGGG)4TT was investigated by polarized Ultraviolet Resonance Raman Scattering (UVRR) at 266 nm. The presence of 40% poly(ethylene glycol) and the so-called "self-crowding" condition were used to induce the hybrid-to-parallel topology transition. Analysis of frequency shifts with temperature showed the role of several functional groups in the topological transitions and provides structural dynamical information. Circular dichroism under similar conditions was used as a reference. UVRR shed light on the effect of intramolecular interactions and of local and environmental dynamics in promoting different G-quadruplex topologies, induced by solution conditions or by temperature changes. Overall, these findings showed the enormous potential of this spectroscopy for G-quadruplex conformational studies.

Huntingtin protein: A new option for fixing the Huntington's disease countdown clock.

Huntington's disease is a dreadful, incurable disorder. It springs from the autosomal dominant mutation in the first exon of the HTT gene, which encodes for the huntingtin protein (HTT) and results in progressive neurodegeneration. Thus far, all the attempted approaches to tackle the mutant HTT-induced toxicity causing this disease have failed. The mutant protein comes with the aberrantly expanded poly-glutamine tract. It is primarily to blame for the build-up of ß-amyloid-like HTT aggregates, deleterious once broadened beyond the critical ∼35-37 repeats threshold. Recent experimental findings have provided valuable information on the molecular basis underlying this HTT-driven neurodegeneration. These findings indicate that the poly-glutamine siding regions and many post-translation modifications either abet or counter the poly-glutamine tract. This review provides an overall, up-to-date insight into HTT biophysics and structural biology, particularly discussing novel pharmacological options to specifically target the mutated protein and thus inhibit its functions and toxicity.

Binding of Harmine Derivatives to DNA: A Spectroscopic Investigation.

Harmine belongs to a group of ß-carboline alkaloids endowed with antitumor properties. Harmine and its derivatives are thought to bind to DNA and interfere with topoisomerase activities. We investigated the base-dependent binding of harmine, and three of its synthetic anticancer-active derivatives to the genomic DNA from calf thymus and two synthetic 20-mer double helices, the poly(dG-dC)·poly(dG-dC) and the poly(dA-dT)·poly(dA-dT), by means of UV-Vis and circular dichroism (CD) spectroscopies. The data show that the DNA binding and stabilising properties of the investigated derivatives are base pair-dependent. These results could be used as a guide to design and develop further bioactive analogues.