Synthesis and Exon-Skipping Properties of a 3'-Ursodeoxycholic Acid-Conjugated Oligonucleotide Targeting DMD Pre-mRNA: Pre-Synthetic versus Post-Synthetic Approach.

Steric blocking antisense oligonucleotides (ASO) are promising tools for splice modulation such as exon-skipping, although their therapeutic effect may be compromised by insufficient delivery. To address this issue, we investigated the synthesis of a 20-mer 2'-OMe PS oligonucleotide conjugated at 3'-end with ursodeoxycholic acid (UDCA) involved in the targeting of human DMD exon 51, by exploiting both a pre-synthetic and a solution phase approach. The two approaches have been compared. Both strategies successfully provided the desired ASO 51 3'-UDC in good yield and purity. It should be pointed out that the pre-synthetic approach insured better yields and proved to be more cost-effective. The exon skipping efficiency of the conjugated oligonucleotide was evaluated in myogenic cell lines and compared to that of unconjugated one: a better performance was determined for ASO 51 3'-UDC with an average 9.5-fold increase with respect to ASO 51.

Octave-spanning coherent perfect absorption in a thin silicon film.

Although optical absorption is an intrinsic materials property, it can be manipulated through structural modification. Coherent perfect absorption increases absorption to 100% interferometrically but is typically realized only over narrow bandwidths using two laser beams with fixed phase relationship. We show that engineering a thin film's photonic environment severs the link between the effective absorption of the film and its intrinsic absorption while eliminating, in principle, bandwidth restrictions. Employing thin aperiodic dielectric mirrors, we demonstrate coherent perfect absorption in a 2 µm thick film of polycrystalline silicon using a single incoherent beam of light at all the resonances across a spectrally flat, octave-spanning near-infrared spectrum, ≈800-1600 nm. Critically, these mirrors have wavelength-dependent reflectivity devised to counterbalance the decline in silicon's intrinsic absorption at long wavelengths.

Analytical model for coherent perfect absorption in one-dimensional photonic structures.

Coherent perfect absorption (CPA) is the phenomenon where a linear system with low intrinsic loss strongly absorbs two incident beams but only weakly absorbs either beam when incident separately. We present an analytical model that captures the relevant physics of CPA in one-dimensional photonic structures. This model elucidates an absorption-mediated interference effect that underlies CPA-an effect that is normally forbidden in Hermitian systems but is allowed when conservation of energy is violated due to the inclusion of loss. By studying a planar cavity model, we identify the optimal mirror reflectivity to guarantee CPA in the cavity at resonances extending in principle over any desired bandwidth. As a concrete example, we design a resonator that produces CPA in a 1-µm-thick layer of silicon over a 200-nm bandwidth in the near-infrared.

Gold-nanoparticle extraction and reversed-electrode-polarity stacking mode combined to enhance capillary electrophoresis sensitivity for conjugated nucleosides and oligonucleotides containing thioether linkers.

We present a capillary electrophoresis method for determining two different C8-conjugated deoxyadenosines, and for oligonucleotides containing them, in which a psoralen or an acridine molecule is bonded to the base via a short alkyl chain containing sulfur ethers at both ends. The sensitivity of the micellar electrokinetic chromatography (MEKC) method was increased by using two preconcentration techniques, micro solid-phase extraction (µSPE) followed by reversed-electrode-polarity stacking mode (REPSM). Variables that affect the efficiency of the extraction in µSPE and preconcentration by REPSM, including the type and volume of extraction nanoparticle, concentration, and injection time, were investigated. Under the optimum conditions, enrichment factors obtained were in the range 360-400. The limits of detection (LODs) at a signal-to-noise ratio of 3 ranged from 2 to 5 nmol L(-1). The relative recoveries of labelled adenosines from water samples were 95-103%. The proposed method provided high enrichment factors and good precision and accuracy with a short analysis time. On the basis of the advantages of simplicity, high selectivity, high sensitivity, and good reproducibility, the proposed method may have great potential for biochemical applications.

Labeling deoxyadenosine for the preparation of functional conjugated oligonucleotides.

Herein we present a versatile synthetic method for the 8-thioalkylation of (deoxy)adenosine with a short carbon linker having on the other side a variety of molecules (psoralen, acridine) and functional groups (alkyne). After conventional protections, the modified adenosine can be phosphytylated and inserted into an oligonucleotide without affecting the standard protocols for supported oligonucleotide synthesis. The hybridization properties of a generic oligonucleotide containing the above conjugated moieties toward both DNA and RNA are evaluated both in the case of a perfectly complementary strand and in the case of a single mismatch. This methodology is suitable for the preparation of several types of derivatives and­through the alkynyl moiety­provides fast access to click-chemistry transformations.

Oligothiophenes as fluorescent markers for biological applications.

This paper summarizes some of our results on the application of oligothiophenes as fluorescent markers for biological studies. The oligomers of thiophene, widely known for their semiconductor properties in organic electronics, are also fluorescent compounds characterized by chemical and optical stability, high absorbance and quantum yield. Their fluorescent emission can be easily modulated via organic synthesis by changing the number of thiophene rings and the nature of side-chains. This review shows how oligothiophenes can be derivatized with active groups such as phosphoramidite, N-hydroxysuccinimidyl and 4-sulfotetrafluorophenyl esters, isothiocyanate and azide by which the (bio)molecules of interest can be covalently bound. This paper also describes how molecules such as oligonucleotides, proteins and even nanoparticles, tagged with oligothiophenes, can be used in experiments ranging from hybridization studies to imaging of fixed and living cells. Finally, a few multilabeling experiments are described.

Can Asymptomatic or Non-Severe SARS-CoV-2 Infection Cause Medium-Term Pulmonary Sequelae in Children?

Pulmonary complications in adults who recovered from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported even in minimally symptomatic patients. In this study, lung ultrasound (LUS) findings and pulmonary function of children who recovered from an asymptomatic or mildly symptomatic SARS-CoV-2 infection were evaluated. We prospectively followed up for at least 30 days patients younger than 18 years who recovered from SARS-CoV-2 infection at the Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan (Italy). All enrolled patients underwent LUS. Airway resistance measured by the interrupter technique test was assessed in subjects aged 4-6 years, whereas forced spirometry and measurement of diffusing capacity of the lungs for carbon monoxide were performed in subjects older than 6 years. To evaluate a possible correlation between pulmonary alterations and immune response to SARS-CoV-2, two semiquantitative enzyme immune assays were used. We enrolled 16 out of 23 eligible children. The median age of enrolled subjects was 7.5 (0.5-10.5) years, with a male to female ratio of 1.7. No subject presented any abnormality on LUS, airway resistance test, forced spirometry, and diffusing capacity of the lungs for carbon monoxide. On the other hand, all subjects presented Ig G against SARS-CoV-2. In contrast in adults, we did not detect any pulmonary complications in our cohort. These preliminary observations suggest that children with an asymptomatic or mildly symptomatic SARS-CoV-2 infection might be less prone to develop pulmonary complications than adults.

Fluoroquinolones as potential photochemotherapeutic agents: covalent addition to guanosine monophosphate.

The triplet aryl cation photochemically generated from fluoroquinolones bearing a fluoro atom at position 8 attacks guanosine monophosphate (k(r) > 10(9) M(-1)s(-1)) and forms covalent adducts. The reaction is a model for the implementation of oxygen-independent photochemotherapy.

Evaluation of the Anticancer Activity of a Bile Acid-Dihydroartemisinin Hybrid Ursodeoxycholic-Dihydroartemisinin in Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy in adults and accounts for 85-90% of all primary liver cancer. Based on the estimation by the International Agency for Research on Cancer in 2018, liver cancer is the fourth leading cause of cancer death globally. Dihydroartemisinin (DHA), the main active metabolite of artemisinin derivatives, is a well-known drug for the treatment of malaria. Previous studies have demonstrated that DHA exhibits antitumor effects toward a variety of human cancers and has a potential for repurposing as an anticancer drug. However, its short half-life is a concern and may limit the application in cancer therapy. We have reported that UDC-DHA, a hybrid of bile acid ursodeoxycholic acid (UDCA) and DHA, is ∼12 times more potent than DHA against a HCC cell line HepG2. In this study, we found that UDC-DHA was also effective against another HCC cell line Huh-7 with an IC50 of 2.16 µM, which was 18.5-fold better than DHA with an IC50 of 39.96 µM. UDC-DHA was much more potent than the combination of DHA and UDCA at 1:1 molar ratio, suggesting that the covalent linkage rather than a synergism between UDCA and DHA is critical for enhancing DHA potency in HepG2 cells. Importantly, UDC-DHA was much less toxic to normal cells than DHA. UDC-DHA induced G0/G1 arrest and apoptosis. Both DHA and UDC-DHA significantly elevated cellular reactive oxygen species generation but with different magnitude and timing in HepG2 cells; whereas only DHA but not UDC-DHA induced reactive oxygen species in Huh-7 cells. Depolarization of mitochondrial membrane potential was detected in both HepG2 and Huh-7 cells and may contribute to the anticancer effect of DHA and UDC-DHA. Furthermore, UDC-DHA was much more stable than DHA based on activity assays and high performance liquid chromatography-MS/MS analysis. In conclusion, UDC-DHA and DHA may exert anticancer actions via similar mechanisms but a much lower concentration of UDC-DHA was required, which could be attributed to a better stability of UDC-DHA. Thus, UDC-DHA could be a better drug candidate than DHA against HCC and further investigation is warranted.

Polydopamine Nanoparticle-Coated Polysulfone Porous Granules as Adsorbents for Water Remediation.

Water purification technologies possibly based on eco-sustainable, low cost, and multifunctional materials are being intensively pursued to resolve the current water scarcity and pollution. In this scenario, polysulfone hollow porous granules (PS-HPGs) prepared from scraps of the industrial production of polysulfone hollow fiber membranes were recently introduced as adsorbents and filtration materials for water and air treatment. Here, we report the functionalization of PS-HPGs with polydopamine (PD) nanoparticles for the preparation of a new versatile and efficient adsorbent material, namely, PSPD-HPGs. The in situ growth of PD under mild alkaline oxidative polymerization allowed us to stably graft PD on polysulfone granules. Enhanced removal efficiency of ofloxacin, an antibiotic drug, with an improvement up to 70% with respect to the pristine PS-HPGs, and removal of Zn(II) and Ni(II) were also observed after PD modification. Remarkably, removal of Cu(II) ions with an efficiency up to 80% was observed for PSPD-HPGs, whereas no adsorption was found for the PD-free precursor. Collectively, these data show that modification with a biocompatible polymer such as PD provides a simple and valuable tool to enlarge the field of application of polysulfone hollow granules for water remediation from both organic and metal cation contaminants.

Dihydroartemisinin-Bile Acid Hybridization as an Effective Approach to Enhance Dihydroartemisinin Anticancer Activity.

A series of hybrid compounds based on natural products-bile acids and dihydroartemisinin-were prepared by different synthetic methodologies and investigated for their in vitro biological activity against HL-60 leukemia and HepG2 hepatocellular carcinoma cell lines. Most of these hybrids presented significantly improved antiproliferative activities with respect to dihydroartemisinin and the parent bile acid. The two most potent hybrids of the series exhibited a 10.5- and 15.4-fold increase in cytotoxic activity respect to dihydroartemisinin alone in HL-60 and HepG2 cells, respectively. Strong evidence that an ursodeoxycholic acid hybrid induced apoptosis was obtained by flow cytometric analysis and western blot analysis.

Targeting MDR1 gene: synthesis and cellular study of modified daunomycin-triplex-forming oligonucleotide conjugates able to inhibit gene expression in resistant cell lines.

Reversal of the multidrug-resistant (MDR) phenotype is very important for chemotherapy success. In fact, the expression of the MDR1 gene-encoded P-glycoprotein (P-gp) actively expels antitumor agents such as daunomycin (DNM) out of the cells, resulting in drug resistance. We show that upon conjugation to triplex-forming oligonucleotides, it is possible to address DNM in resistant cells (MCF7-R and NIH-MDR-G185). The oligonucleotide moiety of the conjugate changes the cellular penetration properties of the antitumor agent that is no more the target of P-gp in resistant cells. We observe an accumulation of conjugated DNM in cells up to 72 h. For more efficient delivery in the cells' nuclei, transfectant agents must be used. In addition, the conjugate recognizes a sequence located in exon 3 of MDR1, and it inhibits its gene expression as measured both by Western blot and by reverse transcription-polymerase chain reaction.

Daunomycin-TFO Conjugates for Downregulation of Gene Expression.

Daunomycin has shown interesting properties as a stabilizing agent for the antigene methodology.This approach consists of targeting a polypurine region of a given gene, with a triplex formingoligonucleotide (TFO), realizing a triple helix complex (triplex), with the aim of down-regulatinggene expression. This chapter describes the basic principles of the triplex approach, the chemistry underliningthe binding of daunomycin to oligonucleotides, and some results of gene-inhibition obtained with daunomycin-TFOconjugates with different targets.

Gaining an insight into the photoreactivity of a drug in a protein environment: a case study on nalidixic acid and serum albumin.

The binding of nalidixic acid (NA) with human and bovine serum albumin (HSA and BSA) in buffer solution at pH 7.4 was investigated using circular dichroism (CD), UV absorption and fluorescence spectroscopy. Global analysis of multiwavelength spectroscopic data afforded the equilibrium constants of the most stable noncovalent drug/protein adducts of 1:1 and 2:1 stoichiometry and their individual CD, UV absorption, and fluorescence spectra. The primary binding site of the drug was located in subdomain IIIA (Sudlow Site II), whereas the secondary one was assigned to subdomain IIA. Conformational and CD calculations afforded the binding geometries. In the complexes, the fluorescence of the protein was strongly quenched by energy transfer and that of the drug was suppressed by electron transfer. Laser flash photolysis at 355 nm evidenced the formation of a radical pair consisting of a tyroxyl radical (lambdamax = 410 nm) and a reduced nalidixate anion radical NA(2-)* (lambdamax = 640 nm) with quantum yield of 0.4-0.5. Strong evidence was obtained that the process that involves Tyr411 in HSA (Tyr409 in BSA). A further transient with lambdamax approximately 780 nm observed in HSA was attributed to oxidation of the -(S200-S246)- bridge upon electron transfer to NA(-)*. Decay of the confined radical pairs occurred with rates approximately 10(7) s(-1). Formation of covalent drug-protein adducts in mixtures irradiated at lambdairr> 324 nm was proved using HPLC with fluorescence detection.

Growth inhibition and apoptosis induced by daunomycin-conjugated triplex-forming oligonucleotides targeting the c-myc gene in prostate cancer cells.

Covalent attachment of intercalating agents to triplex-forming oligonucleotides (TFOs) is a promising strategy to enhance triplex stability and biological activity. We have explored the possibility to use the anticancer drug daunomycin as triplex stabilizing agent. Daunomycin-conjugated TFOs (dauno-TFOs) bind with high affinity and maintain the sequence-specificity required for targeting individual genes in the human genome. Here, we examined the effects of two dauno-TFOs targeting the c-myc gene on gene expression, cell proliferation and survival. The dauno-TFOs were directed to sequences immediately upstream (dauno-GT11A) and downstream (dauno-GT11B) the major transcriptional start site in the c-myc gene. Both dauno-TFOs were able to down-regulate promoter activity and transcription of the endogenous gene. Myc-targeted dauno-TFOs inhibited growth and induced apoptosis of prostate cancer cells constitutively expressing the gene. Daunomycin-conjugated control oligonucleotides with similar sequences had only minimal effects, confirming that the activity of dauno-TFOs was sequence-specific and triplex-mediated. To test the selectivity of dauno-TFOs, we examined their effects on growth of normal human fibroblasts, which express low levels of c-myc. Despite their ability to inhibit c-myc transcription, both dauno-TFOs failed to inhibit growth of normal fibroblasts at concentrations that inhibited growth of prostate cancer cells. In contrast, daunomycin inhibited equally fibroblasts and prostate cancer cells. Thus, daunomycin per se did not contribute to the antiproliferative activity of dauno-TFOs, although it greatly enhanced their ability to form stable triplexes at the target sites and down-regulate c-myc. Our data indicate that dauno-TFOs are attractive gene-targeting agents for development of new cancer therapeutics.

Anti-cancer activity of di- and tri-organotin(IV) compounds with D-(+)-Galacturonic acid on human tumor cells.

We have compared the anti-proliferative activity in vitro, of R2SnGala (1-3) [R = Me, n-Bu, Ph] and novel R3SnGala (4, 5) [R = Me, n-Bu] with D-(+)-Galacturonic acid [HGala; Galaq-, q = (2) and (1) for R2SnGala and R3SnGala, respectively] compounds, towards human tumor cell lines of intestinal carcinoma (HCT-116) and breast adenocarcinoma (MCF-7). The new synthesized 4 and 5 compounds were characterized, in solution, by 1H, 13C and 119Sn NMR, that showed that HGala acts as monoanionic moiety and evidenced the dynamic behavior of the compounds, due to inter-conversions involving the anomeric carbon atom of the ligand. Cell viability, apoptosis induction and cell cycle distribution were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and flow cytometry, respectively. The cytotoxicity of the compounds, in the micro-submicromolar range, changed in the order of the organotin(IV) moieties, according to 5 > 3 > 2, while 1 and 4, containing MenSn(IV) (n = 2,3) moieties, were ineffective. Compound 5 showed peculiar cytotoxic effects. It did not cause time dependent inhibition of cell growth nor accumulated into the cells. Cell death induced by the active 2, 3, and 5, was shown to be apoptotic by measuring the exposure of phosphatidylserine to the outer membrane and the loss of mitochondrial potential. All the cytotoxic compounds induced an accumulation of cells in the subG0/G1phase, while only 2 and 3 perturbed the cell cycle confining viable cells in G0/G1phase. Finally, none of the compounds investigated affected the viability of normal intestinal or liver cells, indicating selectivity towards tumor cells.

Omni-resonant optical micro-cavity.

Optical cavities transmit light only at discrete resonant frequencies, which are well-separated in micro-structures. Despite attempts at the construction of planar 'white-light cavities', the benefits accrued upon optically interacting with a cavity - such as resonant field buildup - have remained confined to narrow linewidths. Here, we demonstrate achromatic optical transmission through a planar Fabry-Pérot micro-cavity via angularly multiplexed phase-matching that exploits a bio-inspired grating configuration. By correlating each wavelength with an appropriate angle of incidence, a continuous spectrum resonates and the micro-cavity is rendered transparent. The locus of a single-order 0.7-nm-wide resonance is de-slanted in spectral-angular space to become a 60-nm-wide achromatic resonance spanning multiple cavity free-spectral-ranges. The result is an 'omni-resonant' planar micro-cavity in which light resonates continuously over a broad spectral span. This approach severs the link between the resonance bandwidth and the cavity-photon lifetime, thereby promising resonant enhancement of linear and nonlinear optical effects over broad bandwidths in ultrathin devices.

DNA binding and antigene activity of a daunomycin-conjugated triplex-forming oligonucleotide targeting the P2 promoter of the human c-myc gene.

Triplex-forming oligonucleotides (TFO) that bind DNA in a sequence-specific manner might be used as selective repressors of gene expression and gene-targeted therapeutics. However, many factors, including instability of triple helical complexes in cells, limit the efficacy of this approach. In the present study, we tested whether covalent linkage of a TFO to daunomycin, which is a potent DNA-intercalating agent and anticancer drug, could increase stability of the triple helix and activity of the oligonucleotide in cells. The 11mer daunomycin-conjugated GT (dauno-GT11) TFO targeted a sequence upstream of the P2 promoter, a site known to be critical for transcription of the c-myc gene. Band-shift assays showed that the dauno-GT11 formed triplex DNA with enhanced stability compared to the unmodified TFO. Band shift and footprinting experiments demonstrated that binding of dauno-GT11 was highly sequence-specific with exclusive binding to the 11 bp target site in the c-myc promoter. The daunomycin-conjugated TFO inhibited transcription in vitro and reduced c-myc promoter activity in prostate and breast cancer cells. The daunomycin-conjugated TFO was taken up by cells with a distinctive intracellular distribution compared to free daunomycin. However, cationic lipid-mediated delivery was required for enhanced cellular uptake, nuclear localization and biological activity of the TFO in cells. Dauno-GT11 reduced transcription of the endogenous c-myc gene in cells, but did not affect expression of non-target genes, such as ets-1 and ets-2, which contained very similar target sequences in their promoters. Daunomycin-conjugated control oligonucleotides unable to form triplex DNA with the target sequence did not have any effect in these assays, indicating that daunomycin was not directly responsible for the activity of daunomycin-conjugated TFO. Thus, attachment of daunomycin resulted in increased triplex stability and biological activity of the 11mer GT-rich TFO without compromising its specificity. These results encourage further testing of this approach to develop novel antigene therapeutics.

NO Photoreleaser-Deoxyadenosine and -Bile Acid Derivative Bioconjugates as Novel Potential Photochemotherapeutics.

This contribution reports the synthesis of some novel bioconjugates with anticancer activity and able to release nitric oxide (NO) under visible light excitation. The 4-nitro-2-(trifluoromethyl)aniline derivative, a suitable NO photodonor, was conjugated with 2'-deoxyadenosine and urso- and cheno-deoxycholic acid derivatives, through a thioalkylic chain or the 4-alkyl-1,2,3-triazole moiety. Photochemical experiments demonstrated the effective release of NO from 2'-deoxyadenosine and ursodeoxycholic acid conjugates under the exclusive control of visible light inputs. Studies for the in vitro antiproliferative activity against leukemic K562 and colon carcinoma HCT116 cell lines are reported for all the compounds as well as a case study of photocytotoxicity against HCT116.