Context-Responsive Nanoparticle Derived from Synthetic Zwitterionic Ionizable Phospholipids in Targeted CRISPR/Cas9 Therapy for Basal-like Breast Cancer.

The majority of triple negative breast cancers (TNBCs) are basal-like breast cancers (BLBCs), which tend to be more aggressive, proliferate rapidly, and have poor clinical outcomes. A key prognostic biomarker and regulator of BLBC is the Forkhead box C1 (FOXC1) transcription factor. However, because of its functional placement inside the cell nucleus and its structural similarity with other related proteins, targeting FOXC1 for therapeutic benefit, particularly for BLBC, continues to be difficult. We envision targeted nonviral delivery of CRISPR/Cas9 plasmid toward the efficacious knockdown of FOXC1. Keeping in mind the challenges associated with the use of CRISPR/Cas9 in vivo, including off-targeting modifications, and effective release of the cargo, a nanoparticle with context responsive properties can be designed for efficient targeted delivery of CRISPR/Cas9 plasmid. Consequently, we have designed, synthesized, and characterized a zwitterionic amino phospholipid-derived transfecting nanoparticle for delivery of CRISPR/Cas9. The construct becomes positively charged only at low pH, which encourages membrane instability and makes it easier for nanoparticles to exit endosomes. This has enabled effective in vitro and in vivo downregulation of protein expression and genome editing. Following this, we have used EpCAM aptamer to make the system targeted toward BLBC cell lines and to reduce its off-target toxicity. The in vivo efficacy, biodistribution, preliminary pharmacokinetics, and biosafety of the optimized targeted CRISPR nanoplatform is then validated in a rodent xenograft model. Overall, we have attempted to knockout the proto-oncogenic FOXC1 expression in BLBC cases by efficient delivery of CRISPR effectors via a context-responsive nanoparticle delivery system derived from a designer lipid derivative. We believe that the nonviral approach for in vitro and in vivo delivery of CRISPR/Cas9 targeted toward FOXC1, studied herein, will greatly emphasize the therapeutic regimen for BLBC.

Expanding the chemical space of enol silyl ethers: catalytic dicarbofunctionalization enabled by iron catalysis.

Enol silyl ethers are versatile, robust, and readily accessible substrates widely used in chemical synthesis. However, the conventional reactivity of these motifs has been limited to classical two electron (2-e) enolate-type chemistry with electrophilic partners or as radical acceptors in one electron (1-e) reactivity leading, in both cases, to exclusive α-monofunctionalization of carbonyls. Herein we describe a mild, fast, and operationally simple one-step protocol that combines readily available fluoroalkyl halides, silyl enol ethers, and, for the first time, hetero(aryl) Grignard reagents to promote selective dicarbofunctionalization of enol silyl ethers. From a broader perspective, this work expands the synthetic utility of enol silyl ethers and establishes bisphosphine-iron catalysis as enabling technology capable of orchestrating selective C-C bond formations with short-lived α-silyloxy radicals with practical implications towards sustainable chemical synthesis.

Ensemble and single-particle level fluorescent fine-tuning of carbon dots via positional changes of amines toward "supervised" oral microbiome sensing.

Significance: Carbon dots (CDs) have attracted a host of research interest in recent years mainly due to their unique photoluminescence (PL) properties that make them applicable in various biomedical areas, such as imaging and image-guided therapy. However, the real mechanism underneath the PL is a subject of wide controversy and can be investigated from various angles. Aim: Our work investigates the effect of the isomeric nitrogen position as the precursor in the synthesis of CDs by shedding light on their photophysical properties on the single particles and ensemble level. Approach: To this end, we adopted five isomers of diaminopyridine (DAP) and urea as the precursors and obtained CDs during a hydrothermal process. The various photophysical properties were further investigated in depth by mass spectroscopy. CD molecular frontier orbital analyses aided us in justifying the fluorescence emission profile on the bulk level as well as the charge transfer processes. As a result of the varying fluorescent responses, we indicate that these particles can be utilized for machine learning (ML)-driven sensitive detection of oral microbiota. The sensing results were further supported by density functional theoretical calculations and docking studies. Results: The generating isomers have a significant effect on the overall photophysical properties at the bulk/ensembled level. On the single-particle level, although some of the photophysical properties such as average intensity remained the same, the overall differences in brightness, photo-blinking frequency, and bleaching time between the five samples were conceived. The various photophysical properties could be explained based on the different chromophores formed during the synthesis. Overall, an array of CDs was demonstrated herein to achieve ∼100% separation efficacy in segregating a mixed oral microbiome culture in a rapid (<0.5 h), high-throughput manner with superior accuracy. Conclusions: We have indicated that the PL properties of CDs can be regulated by the precursors' isomeric position of nitrogen. We emancipated this difference in a rapid method relying on ML algorithms to segregate the dental bacterial species as biosensors.

Small Molecule NIR-II Dyes for Switchable Photoluminescence via Host -Guest Complexation and Supramolecular Assembly with Carbon Dots.

Small molecular NIR-II dyes are highly desirable for various biomedical applications. However, NIR-II probes are still limited due to the complex synthetic processes and inadequate availability of fluorescent core. Herein, the design and synthesis of three small molecular NIR-II dyes are reported. These dyes can be excited at 850-915 nm and emitted at 1280-1290 nm with a large stokes shift (≈375 nm). Experimental and computational results indicate a 2:1 preferable host-guest assembly between the cucurbit[8]uril (CB) and dye molecules. Interestingly, the dyes when self-assembled in presence of CB leads to the formation of nanocubes (≈200 nm) and exhibits marked enhancement in fluorescence emission intensity (Switch-On). However, the addition of red carbon dots (rCDots, ≈10 nm) quenches the fluorescence of these host-guest complexes (Switch-Off) providing flexibility in the user-defined tuning of photoluminescence. The turn-ON complex found to have comparable quantum yield to the commercially available near-infrared fluorophore, IR-26. The aqueous dispersibility, cellular and blood compatibility, and NIR-II bioimaging capability of the inclusion complexes is also explored. Thus, a switchable fluorescence behavior, driven by host-guest complexation and supramolecular self-assembly, is demonstrated here for three new NIR-II dyes.

Fe-Catalyzed dicarbofunctionalization of electron-rich alkenes with Grignard reagents and (fluoro)alkyl halides.

An iron-catalyzed regioselective dicarbofunctionalization of electron-rich alkenes is described. In particular, aryl- and alkyl vinyl ethers are used as effective linchpins to couple alkyl or (fluoro)alkyl halides and sp2-hybridized Grignard nucleophiles. Preliminary results demonstrate the ability to engage thioethers as linchpins and control enantioselectivity in these transformations, an area which is largely unexplored in iron-catalyzed three-component cross-coupling reactions.

Luminescence switching in polymerically confined carbon nanoparticles triggered by UV-light.

Photo-caged carbon nanoparticles (CNPs) that are non-luminescent under typical microscopic illumination but can be activated by UV light have been synthesized in this work. Negatively charged "bare" CNPs with high luminescence can lose their photoluminescence (PL) when they are chemically crosslinked to a monomer and subsequently polymerized to form an intra-particulate "caged" network at the nanoscale surface. These caged particles could regain their PL emission upon UV irradiation for a sustained period (∼24 h) resulting in the photolytic cleavage of the polymer network, thus, freeing the nanoscale surface of CNPs, ultimately resulting in six-fold emission enhancement. This reversible "on-off-on" PL switching process was verified by spectroscopic techniques. We successfully demonstrated in this work that CNPs can be switched reversibly between fluorescent and non-fluorescent states by irradiation with light. These results further substantiate that the origin of PL in CNPs is a surface phenomenon and highly dependent on their nanoscale coverage.

Function-adaptive clustered nanoparticles reverse Streptococcus mutans dental biofilm and maintain microbiota balance.

Dental plaques are biofilms that cause dental caries by demineralization with acidogenic bacteria. These bacteria reside inside a protective sheath which makes any curative treatment challenging. We propose an antibiotic-free strategy to disrupt the biofilm by engineered clustered carbon dot nanoparticles that function in the acidic environment of the biofilms. In vitro and ex vivo studies on the mature biofilms of Streptococcus mutans revealed >90% biofilm inhibition associated with the contact-mediated interaction of nanoparticles with the bacterial membrane, excessive reactive oxygen species generation, and DNA fragmentation. An in vivo examination showed that these nanoparticles could effectively suppress the growth of S. mutans. Importantly, 16S rRNA analysis of the dental microbiota showed that the diversity and richness of bacterial species did not substantially change with nanoparticle treatment. Overall, this study presents a safe and effective approach to decrease the dental biofilm formation without disrupting the ecological balance of the oral cavity.

Oligodots: Structurally Defined Fluorescent Nanoprobes for Multiscale Dual-Color Imaging in Vitro and in Vivo.

Nanoscale fluorescent probes are of great importance due to their capabilities for imaging on multiscale. Herein, we report the first synthesis of structurally well-defined nanoparticulate "oligodots" developed for multicolor imaging in vitro and in vivo. These nanoparticles are prepared via condensation and curing reactions where the engineering of the solvent results in the nanoparticles with green (λem = 550 nm) and red (λem = 650 nm) emission range. Differences found in the photophysical properties have been attributed to variations in oligomeric compositions produced during the synthesis as was corroborated by extensive physicochemical characterizations. Specifically, mass spectroscopy provided a picture of the formed species during the synthesis. The feasibility of the oligodots for multicolor imaging is demonstrated both in vitro and in vivo. The red-emitting oligodot is employed for dynamic whole-body imaging in mice. It is envisioned that oligodots would enable multicolor imaging of various biomarkers in complex diseases such as cancer where numerous molecular and metabolic phenotypes work in concert in their emergence.

Biodegradable Biliverdin Nanoparticles for Efficient Photoacoustic Imaging.

Photoacoustic imaging has emerged as a promising imaging platform with a high tissue penetration depth. However, biodegradable nanoparticles, especially those for photoacoustic imaging, are rare and limited to a few polymeric agents. The development of such nanoparticles holds great promise for clinically translatable diagnostic imaging with high biocompatibility. Metabolically digestible and inherently photoacoustic imaging probes can be developed from nanoprecipitation of biliverdin, a naturally occurring heme-based pigment. The synthesis of nanoparticles composed of a biliverdin network, cross-linked with a bifunctional amine linker, is achieved where spectral tuning relies on the choice of reaction media. Nanoparticles synthesized in water or water containing sodium chloride exhibit higher absorbance and lower fluorescence compared to nanoparticles synthesized in 2-(N-morpholino)ethanesulfonic acid buffer. All nanoparticles display high absorbance at 365 and 680 nm. Excitation at near-infrared wavelengths leads to a strong photoacoustic signal, while excitation with ultraviolet wavelengths results in fluorescence emission. In vivo photoacoustic imaging experiments in mice demonstrated that the nanoparticles accumulate in lymph nodes, highlighting their potential utility as photoacoustic agents for sentinel lymph node detection. The biotransformation of these agents was studied using mass spectroscopy, and they were found to be completely biodegraded in the presence of biliverdin reductase, a ubiquitous enzyme found in the body. Degradation of these particles was also confirmed in vivo. Thus, the nanoparticles developed here are a promising platform for biocompatible biological imaging due to their inherent photoacoustic and fluorescent properties as well as their complete metabolic digestion.

Enzyme-catalysed biodegradation of carbon dots follows sequential oxidation in a time dependent manner.

Carbon dots (CDs) have recently garnered significant attention owing to their excellent luminescence properties, thereby demonstrating a variety of applications in in vitro and in vivo imaging. Understanding the long-term metabolic fate of these agents in a biological environment is the focus of this work. Here we show that the CDs undergo peroxide catalysed degradation in the presence of lipase. Our results indicate that differently charged CD species exhibit unique degradation kinetics upon being subjected to enzyme oxidation. Furthermore, this decomposition correlates with the relative accessibility of the enzymatic molecule. Using multiple physico-chemical characterization studies and molecular modelling, we confirmed the interaction of passivating surface abundant molecules with the enzyme. Finally, we have identified hydroxymethyl furfural as a metabolic by-product of the CDs used here. Our results indicate the possibility and a likely mechanism for complete CD degradation in living systems that can pave the way for a variety of biomedical applications.

Copper-Catalyzed Syntheses of Pyrene-Pyrazole Pharmacophores and Structure Activity Studies for Tubulin Polymerization.

Tubulin polymerization is critical in mitosis process, which regulates uncontrolled cell divisions. Here, we report a new class of pyrene-pyrazole pharmacophore (PPP) for targeting microtubules. Syntheses of seven pyrenyl-substituted pyrazoles with side-chain modification at N-1 and C-3 positions of the pyrazole ring were accomplished from alkenyl hydrazones via C-N dehydrogenative cross-coupling using copper catalyst under aerobic condition. Tubulin polymerization with PPPs was investigated using docking and biological tools to reveal that these ligands are capable of influencing microtubule polymerization and their interaction with α-, ß-tubulin active binding sites, which are substituent specific. Furthermore, cytotoxicity response of these PPPs was tested on cancer cells of different origin, such as MCF-7, MDA-MB231, and C32, and also noncancerous normal cells, such as MCF-10A. All newly synthesized PPPs showed excellent anticancer activities. The anticancer activities and half-maximal inhibitory concentration (IC50) values of all PPPs across different cancer cell lines (MCF-7, MDA-MB231, and C32) have been demonstrated. 1,3-Diphenyl-5-(pyren-1-yl)-1H-pyrazole was found to be best among all other PPPs in killing significant population of all of the cancerous cell with IC50 values 1 ± 0.5, 0.5 ± 0.2, and 5.0 ± 2.0 µM in MCF-7, MDA-MB231, and C32 cells, respectively.

Carbon dots with induced surface oxidation permits imaging at single-particle level for intracellular studies.

For robust single particle optical detection, a high sensitivity in photoluminescence (PL) of Carbon Dots (CDs) must be achieved. PL sensitivity can be successfully correlated with their surface chemistry but requires high synthetic control without altering their basic surface properties. Here we describe conditions for the controlled synthesis of CDs that resulted in a PL sensitivity at the single-particle level. We report that a stoichiometric catalyst N-methyl morpholine-N-oxide (NMMO) can be used as a 'sacrificial' single additive to aid nanoscale surface oxidation. A 24 h NMMO-mediated oxidation increased coverage of oxidized nanoscale surface 3% to 20.9%. NMMO-oxidized CDs (CD-NMMOs) display superior particle brightness, as evidenced by the increase of light absorbance and an enhancement of quantum yield which is characterized by a series of physicochemical and biophysical experiments. We also demonstrate that CD-NMMOs is well suited for intracellular and single-particle imaging.

Electrically-receptive and thermally-responsive paper-based sensor chip for rapid detection of bacterial cells.

Although significant technological advancements have been made in the development of analytical biosensor chips for detecting bacterial strains (E. coli, S. Mutans and B. Subtilis), critical requirements i.e. limit of detection (LOD), fast time of response, ultra-sensitivity with high reproducibility and good shelf-life with robust sensing capability have yet to be met within a single sensor chip. In order to achieve these criteria, we present an electrically-receptive thermally-responsive (ER-TR) sensor chip comprised of simple filter paper used as substrate coated with composite of poly(N-isopropylacrylamide) polymer (PNIPAm) - graphene nanoplatelet (GR) followed by evaporation of Au electrodes for capturing both Gram-positive (S. mutans and B. subtilis) and Gram-negative (E. coli) bacterial cells in real-time. Autoclave water, tap water, lake water and milk samples were tested with ER-TR chip with and without bacterial strains at varying concentration range 101-105 cells/mL. The sensor was integrated with in-house built printed circuit board (PCB) to transmit/receive electrical signals. The interaction of E. coli, S. mutans and B. subtilis cells with fibers of PNIPAm-GR resulted in a change of electrical resistance and the readout was monitored wirelessly in real-time using MATLAB algorithm. Finally, prepared ER-TR chip exhibited the reproducibility of 85-97% with shelf-life of up to four weeks after testing with lake water sample.

Revisiting Polyarenes and Related Molecules: An Update of Synthetic Approaches and Structure-Activity-Mechanistic Correlation for Carcinogenesis.

A major proportion of basic cause for human cancer has been linked to widespread environmental pollutants including analogs of polyarenes. Search of an effective therapy can be started with the understanding of the generation of such "carcinogens" and their biological interactions. This review is to discuss the syntheses, structural activities, mechanistic and biological studies of polyarenes such as polycyclic aromatic hydrocarbons (PAHs), polycyclic azaarenes (PAAs) and their thia-analogs (PASH). It also summarizes the mechanism of mutagenicity and tumorigenicity via metabolic interventions producing diol epoxide complexes and eventually formation of DNA adducts. It suggests that inhibition of oxidative reactions and formation of diols and epoxides and unspecific intracellular activation of cytochrome P450 enzymes could be approaches in therapy against such mutagenicity and tumorigenicity. Thus, this review reflects that understanding of molecular mechanisms and activations along with a clinical and translational medicine approach would require achieving both prevention and treatment of this atrocity.

Aerobic Metal-Free Dioxygenation of Alkenes with tert-Butyl Nitrite and N-Hydroxylamines.

Metal-free dioxygenation of alkenes with tert-butyl nitrite and N-hydroxylamines (N-hydroxyphthalimide, N-hydroxybenzotriazole, and N-hydroxysuccinimide) is described to produce ß-aminoxy nitrate esters using air as the oxidant. These organic nitrates can be readily converted into 1,2-diols and 1,2-diketone with broad substrate scope and functional group diversity.

Iron(III)-catalyzed aerobic dioxygenation of styrenes using N-hydroxyphthalimide and N-hydroxybenzotriazole.

Iron-catalyzed dioxygenation of alkenes in air using N-hydroxyphthalimide (NHPI) and N-hydroxybenzotriazole (HOBt) is described at room temperature. The products can be readily converted into 1,2-vicinal diols using PPh3 and Mo(CO)6. The use of Fe(NO3)3·9H2O as the catalyst, mild reaction conditions and the selectivity are the significant practical features. The radical scavenger experiment suggests that the reaction proceeds via a radical intermediate. The synthetic and mechanistic aspects have been presented.

Synthesis of Functionalized Pyrazoles via Vanadium-Catalyzed C-N Dehydrogenative Cross-Coupling and Fluorescence Switch-On Sensing of BSA Protein.

Vanadium-catalyzed C-N dehydrogenative cross-coupling of alkenyl hydrazones leading to functionalized pyrazoles is described in a 1:1 mixture of toluene/H2O using air as the terminal oxidant. Significant practical features include use of the commercial nontoxic VOSO4 as a recyclable catalyst, mild reaction conditions, scalability, and the broad substrate scope. Some of the product pyrazoles exhibit interesting photophysical properties. Fluorescence light-up sensing of BSA protein by one of the pyrazoles is also highlighted.

Iron(III)-Mediated Radical Nitration of Bisarylsulfonyl Hydrazones: Synthesis of Bisarylnitromethyl Sulfones.

Iron(III)-mediated radical nitration of bisarylsulfonyl hydrazones is described. In this protocol, the nontoxic and inexpensive Fe(NO3)3·9H2O plays a dual role as catalyst as well as nitro source. The mild conditions, broad substrate scope, and the functional group compatibility are the significant features. The reaction pathway has been demonstrated using DFT calculations, and the products can be subsequently converted into oximes using SnCl2·2H2O in high yields.

Copper(II)-catalyzed direct dioxygenation of alkenes with air and N-hydroxyphthalimide: synthesis of ß-keto-N-alkoxyphthalimides.

Copper(II)-catalyzed direct dioxygenation of alkenes using air and a simple N-hydroxyphthalimide leading to ß-keto-N-alkoxyphthalimides has been developed. The reaction system is mild, efficient, and effective at room temperature with broad substrate scope and substantial steric hindrance. The radical-trapping and (18)O-labeling experiments have been demonstrated.