DNA Origami in the Quest for Membrane Piercing.

The tool kit for label-free single-molecule sensing, nucleic acid sequencing (DNA, RNA and protein) and other biotechnological applications has been significantly broadened due to the wide range of available nanopore-based technologies. Currently, various sources of nanopores, including biological, fabricated solid-state, hybrid and recently de novo chemically synthesized ion-like channels have put in use for rapid single-molecule sensing of biomolecules and other diagnostic applications. At length scales of hundreds of nanometers, DNA nanotechnology, particularly DNA origami-based devices, enables the assembly of complex and dynamic 3-dimensional nanostructures, including nanopores with precise control over the size/shape. DNA origami technology has enabled to construct nanopores by DNA alone or hybrid architects with solid-state nanopore devices or nanocapillaries. In this review, we briefly discuss the nanopore technique that uses DNA nanotechnology to construct such individual pores in lipid-based systems or coupled with other solid-state devices, nanocapillaries for enhanced biosensing function. We summarize various DNA-based design nanopores and explore the sensing properties of such DNA channels. Apart from DNA origami channels we also pointed the design principles of RNA nanopores for peptide sensing applications.

Nano-in-Microparticles for Aerosol Delivery of Antibiotic-Loaded, Fucose-Derivatized, and Macrophage-Targeted Liposomes to Combat Mycobacterial Infections: In Vitro Deposition, Pulmonary Barrier Interactions, and Targeted Delivery.

Nontuberculous mycobacterial infections rapidly emerge and demand potent medications to cope with resistance. In this context, targeted loco-regional delivery of aerosol medicines to the lungs is an advantage. However, sufficient antibiotic delivery requires engineered aerosols for optimized deposition. Here, the effect of bedaquiline-encapsulating fucosylated versus nonfucosylated liposomes on cellular uptake and delivery is investigated. Notably, this comparison includes critical parameters for pulmonary delivery, i.e., aerosol deposition and the noncellular barriers of pulmonary surfactant (PS) and mucus. Targeting increases liposomal uptake into THP-1 cells as well as peripheral blood monocyte- and lung-tissue derived macrophages. Aerosol deposition in the presence of PS, however, masks the effect of active targeting. PS alters antibiotic release that depends on the drug's hydrophobicity, while mucus reduces the mobility of nontargeted more than fucosylated liposomes. Dry-powder microparticles of spray-dried bedaquiline-loaded liposomes display a high fine particle fraction of >70%, as well as preserved liposomal integrity and targeting function. The antibiotic effect is maintained when deposited as powder aerosol on cultured Mycobacterium abscessus. When treating M. abscessus infected THP-1 cells, the fucosylated variant enabled enhanced bacterial killing, thus opening up a clear perspective for the improved treatment of nontuberculous mycobacterial infections.

Co-Delivery of mRNA and pDNA Using Thermally Stabilized Coacervate-Based Core-Shell Nanosystems.

Co-delivery of different species of protein-encoding polynucleotides, e.g., messenger RNA (mRNA) and plasmid DNA (pDNA), using the same nanocarrier is an interesting topic that remains scarcely researched in the field of nucleic acid delivery. The current study hence aims to explore the possibility of the simultaneous delivery of mRNA (mCherry) and pDNA (pAmCyan) using a single nanocarrier. The latter is based on gelatin type A, a biocompatible, and biodegradable biopolymer of broad pharmaceutical application. A core-shell nanostructure is designed with a thermally stabilized gelatin-pDNA coacervate in its center. Thermal stabilization enhances the core's colloidal stability and pDNA shielding effect against nucleases as confirmed by nanoparticle tracking analysis and gel electrophoresis, respectively. The stabilized, pDNA-loaded core is coated with the cationic peptide protamine sulfate to enable additional surface-loading with mRNA. The dual-loaded core-shell system transfects murine dendritic cell line DC2.4 with both fluorescent reporter mRNA and pDNA simultaneously, showing a transfection efficiency of 61.4 ± 21.6% for mRNA and 37.6 ± 19.45% for pDNA, 48 h post-treatment, whereas established commercial, experimental, and clinical transfection reagents fail. Hence, the unique co-transfectional capacity and the negligible cytotoxicity of the reported system may hold prospects for vaccination among other downstream applications.

Spray-dried lactose-leucine microparticles for pulmonary delivery of antimycobacterial nanopharmaceuticals.

Pulmonary delivery of nanocarriers for novel antimycobacterial compounds is challenging because the aerodynamic properties of nanomaterials are sub-optimal for such purposes. Here, we report the development of dry powder formulations for nanocarriers containing benzothiazinone 043 (BTZ) or levofloxacin (LVX), respectively. The intricacy is to generate dry powder aerosols with adequate aerodynamic properties while maintaining both nanostructural integrity and compound activity until reaching the deeper lung compartments. Microparticles (MPs) were prepared using vibrating mesh spray drying with lactose and leucine as approved excipients for oral inhalation drug products. MP morphologies and sizes were measured using various biophysical techniques including determination of geometric and aerodynamic mean sizes, X-ray diffraction, and confocal and focused ion beam scanning electron microscopy. Differences in the nanocarriers' characteristics influenced the MPs' sizes and shapes, their aerodynamic properties, and, hence, also the fraction available for lung deposition. Spay-dried powders of a BTZ nanosuspension, BTZ-loaded silica nanoparticles (NPs), and LVX-loaded liposomes showed promising respirable fractions, in contrast to zirconyl hydrogen phosphate nanocontainers. While the colloidal stability of silica NPs was improved after spray drying, MPs encapsulating either BTZ nanosuspensions or LVX-loaded liposomes showed the highest respirable fractions and active pharmaceutical ingredient loads. Importantly, for the BTZ nanosuspension, biocompatibility and in vitro uptake by a macrophage model cell line were improved even further after spray drying.

Preferential uptake of chitosan-coated PLGA nanoparticles by primary human antigen presenting cells.

Biodegradable polymeric nanoparticles (NP) made from poly (lactid-co-glycolide) acid (PLGA) and chitosan (CS) hold promise as innovative formulations for targeted delivery. Since interactions of such NP with primary human immune cells have not been characterized, yet, here we assessed the effect of PLGA or CS-PLGA NP treatment on human peripheral blood mononuclear cells (PBMC), as well as on monocyte-derived DC (moDC). Amongst PBMC, antigen presenting cells (APC) showed higher uptake of both NP preparations than lymphocytes. Furthermore, moDC internalized CS-PLGA NP more efficiently than PLGA NP, presumably because of receptor-mediated endocytosis. Consequently, CS-PLGA NP were delivered mostly to endosomal compartments, whereas PLGA NP primarily ended up in lysosomes. Thus, CS-PLGA NP confer enhanced delivery to endosomal compartments of APC, offering new therapeutic options to either induce or modulate APC function and to inhibit pathogens that preferentially infect APC.

Hyaluronic acid - dihydroartemisinin conjugate: Synthesis, characterization and in vitro evaluation in lung cancer cells.

In recent years, a great deal of attention has been given towards re-purposing and re-innovating the potential drugs. In this regard, dihydroartemisinin (DHA) has been reported to demonstrate anti-proliferative effects on various cancerous cells viz. breast, liver and lung. However, it is associated with some limitations, such as low bioavailability which is hampered by its poor aqueous solubility and its rapid metabolism in systemic circulation. Therefore, in order to overcome these limitations, we synthesized a novel hyaluronic acid-dihydroartemisinin conjugate in which the hydroxyl group of DHA was covalently linked to carboxylic group of hyaluronic acid (HA). The conjugate was successfully characterized using 1H NMR, Fourier transform infrared spectroscopy (FT-IR) and gel permeation chromatography (GPC). The synthesized conjugate self-assembled into nanoparticles in aqueous solution. The developed nanoparticles were characterized for their average size, zeta potential, Transmission Electron Microscopy (TEM), X-ray Powder Diffraction (XRD) and loading efficiency. The nanoparticles were cytotoxic to lung cancer (A549) cell line which was determined using CCK-8 cell viability assay. This was further supported by Annexin-V-FITC-Propidium iodide apoptosis assay, reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) loss. Conclusively, present findings demonstrate hyaluronic acid conjugates can be used to improve the therapeutic outcomes of anticancer drugs.

Amphiphilic Cargo-Loaded Nanocarrier Enhances Antibiotic Uptake and Perturbs Efflux: Effective Synergy for Mitigation of Methicillin-Resistant Staphylococcus aureus.

A pyridinium-amphiphile-loaded poly(lactic-co-glycolic acid) (PLGA) nanocarrier (C1-PNC) was developed as an adjuvant in order to break the resistance and restore the susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) cells to therapeutic antibiotics. Notably, against a clinical MRSA strain, C1-PNC was found to render 8- and 6-fold decreases in the minimum biofilm eradication concentration (MBEC90 ) of gentamicin and ciprofloxacin, respectively. Mechanistic studies on MRSA planktonic cells revealed that in the case of gentamicin, C1-PNC promotes enhanced cellular uptake of the antibiotic, whereas the propensity of C1-PNC to inhibit efflux pump activity could be leveraged to enhance cellular accumulation of ciprofloxacin, leading to effective killing of MRSA cells. Interestingly, the combinatorial dosing regimen of C1-PNC and the antibiotics was nontoxic to cultured HEK293 cells. This nontoxic amphiphile-loaded nanomaterial holds considerable promise as an adjuvant for antibiotic-mediated alleviation of MRSA biofilms.

A Nonbactericidal Zinc-Complexing Ligand as a Biofilm Inhibitor: Structure-Guided Contrasting Effects on Staphylococcus aureus Biofilm.

Zinc-complexing ligands are prospective anti-biofilm agents because of the pivotal role of zinc in the formation of Staphylococcus aureus biofilm. Accordingly, the potential of a thiosemicarbazone (compound C1) and a benzothiazole-based ligand (compound C4) in the prevention of S. aureus biofilm formation was assessed. Compound C1 displayed a bimodal activity, hindering biofilm formation only at low concentrations and promoting biofilm growth at higher concentrations. In the case of C4, a dose-dependent inhibition of S. aureus biofilm growth was observed. Atomic force microscopy analysis suggested that at higher concentrations C1 formed globular aggregates, which perhaps formed a substratum that favored adhesion of cells and biofilm formation. In the case of C4, zinc supplementation experiments validated zinc complexation as a plausible mechanism of inhibition of S. aureus biofilm. Interestingly, C4 was nontoxic to cultured HeLa cells and thus has promise as a therapeutic anti-biofilm agent. The essential understanding of the structure-driven implications of zinc-complexing ligands acquired in this study might assist future screening regimes for identification of potent anti-biofilm agents.

A near-infrared emissive Al(3+) sensing platform for specific detection in solution, cells and probing DNase activity.

A new tricarbocyanine-based chemosensor exhibited a dramatic Al(3+)-specific fluorescence turn-on response in the near-infrared (NIR) region. The receptor was found to be highly selective towards Al(3+) over other metal ions in physiological condition. The sensor was non-toxic and could thus be employed as an imaging probe for detecting intracellular Al(3+) in live cells. Interestingly, upon interaction with DNA in solution, the L-Al(3+) ensemble rendered tracking of DNase activity in solution through a systematic reduction in the fluorescence emission intensity.

A sole multi-analyte receptor responds with three distinct fluorescence signals: traffic signal like sensing of Al(3+), Zn(2+) and F(-).

A dialdehyde-based multi-analyte sensor renders distinctive emission spectra for Al(3+), Zn(2+) and F(-) ions. The ligand exhibited different types of interactions with these three different ions resulting in the enhancement of fluorescence intensity at three different wavelengths. All the sensing processes were studied in detail by absorption spectroscopy, emission spectroscopy and (1)H-NMR titration experiment. The ligand has the working ability in a wide pH range including the physiological pH. The ligand is non-toxic and amicable for sensing intracellular Al(3+) and Zn(2+) in live HeLa cells.

A zinc complex of a neutral pyridine-based amphiphile: a highly efficient and potentially therapeutic bactericidal material.

The alarming rise in antibiotic-resistant pathogenic bacteria demands a prudent approach in the generation of therapeutic antibacterials. The present study illustrates the development of a potent amphiphilic bactericidal material tailored to leverage interactions with metal-reactive groups (MRGs) present in the bacterial cell surface envelope. Complexation of Zn(ii) with a neutral pyridine-based synthetic amphiphile (C1) generated the cationic C1-Zn, which exhibited manyfold higher membrane-directed bactericidal activity compared to the neutral C1, or the cationic amphiphile bearing two pyridinium head groups (C2). The relevance of MRGs in C1-Zn-bacteria interactions was validated by amphiphile-bacteria binding studies and metal protection assays performed with Mg(ii). C1-Zn retained its bactericidal activity even in simulated gastric fluid (SGF) and the enhanced membrane-directed bactericidal activity of C1-Zn could be garnered in adjuvant applications to increase the efficacy of the therapeutic antibiotic erythromycin. Given the relevance of Zn(ii) in S. aureus biofilm formation, the antibiofilm potential of the amphiphile C1 realized through Zn(ii) complexation could be demonstrated. The lack of resistance in target bacteria coupled with a favorable therapeutic index (IC50/MIC) and non-toxic nature hold significant implications for C1-Zn as a potential antibacterial therapeutic material.

Biocompatible nanocarrier fortified with a dipyridinium-based amphiphile for eradication of biofilm.

Annihilation of bacterial biofilms is challenging owing to their formidable resistance to therapeutic antibiotics and thus there is a constant demand for development of potent antibiofilm agents that can abolish established biofilms. In the present study, the activity of a dipyridinium-based cationic amphiphile (compound 1) against established bacterial biofilms and the subsequent development of a compound 1-loaded nanocarrier for potential antibiofilm therapy are highlighted. Solution-based assays and microscopic analysis revealed the antagonistic effect of compound 1 on biofilms formed by Staphylococcus aureus MTCC 96 and Pseudomonas aeruginosa MTCC 2488. In combination studies, compound 1 could efficiently potentiate the action of tobramycin and gentamicin on P. aeruginosa and S. aureus biofilm, respectively. A human serum albumin (HSA)-based nanocarrier loaded with compound 1 was generated, which exhibited sustained release of compound 1 at physiological pH. The compound 1-loaded HSA nanocarrier (C1-HNC) displayed the signature membrane-directed activity of the amphiphile on target bacteria, efficiently eliminated established bacterial biofilms, and was observed to be nontoxic to a model human cell line. Interestingly, compound 1 as well as the amphiphile-loaded HSA nanocarrier could eradicate established S. aureus biofilm from the surface of a Foley's urinary catheter. On the basis of its biocompatibility and high antibiofilm activity, it is conceived that the amphiphile-loaded nanocarrier may hold potential in antibiofilm therapy.

A novel chemosensor with visible light excitability for sensing Zn2+ in physiological medium and in HeLa cells.

In the present study a novel imine-hydrazone based fluorescent chemosensor () for efficient and selective sensing of Zn(2+) over other biologically important metal ions under physiological conditions is reported. An enhancement in fluorescence emission intensity of the developed probe with a red shift of ∼25 nm was observed for Zn(2+), whereas other metal ions failed to reveal any significant change in the emission spectra. Interestingly, the receptor functioned under completely physiological conditions (99.7% HEPES buffer) and has visible light excitability. Sensing of Zn(2+) was investigated in detail by absorption spectroscopy, emission spectroscopy, DFT calculation, (1)H-NMR titration experiment and ESI-MS experiment. The association constant between and Zn(2+) was found to be 5.58 × 10(5) M(-1). The receptor could detect as low as 69 ppb Zn(2+). Sensing of Zn(2+) is proposed through switch-on of intramolecular charge transfer (ICT) and chelation enhanced fluorescence (CHEF) processes after the introduction of Zn(2+) into the free ligand. The developed receptor was non-toxic and rendered intracellular sensing of Zn(2+) in HeLa cells through fluorescence imaging studies.

A prospective antibacterial for drug-resistant pathogens: a dual warhead amphiphile designed to track interactions and kill pathogenic bacteria by membrane damage and cellular DNA cleavage.

A rationally designed bactericidal amphiphile acts on compelling targets and has the potential to disarm resistance development in pathogenic bacteria.

Amphiphile-mediated enhanced antibiotic efficacy and development of a payload nanocarrier for effective killing of pathogenic bacteria.

Synthetic amphiphiles have emerged as potent bactericidal scaffolds owing to their high propensity to interact with bacterial cells and a membrane-directed mode of action, which is likely to overcome resistance development in pathogenic bacteria. In this study, we highlighted a membrane-acting quinolinium-based cationic amphiphile (compound 1) as an adjuvant for antibiotic-mediated eradication of pathogenic bacteria and demonstrated the generation of an amphiphile-loaded nanocarrier for potential antibacterial therapy. Treatment of Gram-negative pathogenic bacteria E. coli MTCC 433 and P. aeruginosa MTCC 2488 with 1 resulted in significant augmentation of the activity of erythromycin and a decrease in the minimum inhibitory concentration of the antibiotic. Interestingly, 1 promoted large-scale eradication of P. aeruginosa MTCC 2488 biofilm and could also enhance the anti-biofilm activity of tobramycin in combination. For potential therapeutic applications, a 1-loaded bovine serum albumin-based nanocarrier was developed, which exhibited sustained release of 1 both in physiological and acidic pH and the released amphiphile displayed antibacterial as well as anti-biofilm activities. Interestingly, the nanocarrier also displayed the signature membrane-directed activity of 1 against tested pathogenic bacteria. The high bactericidal and anti-biofilm activities in conjunction with a lack of cytotoxic effect on HT-29 human cell lines enhance the merit of the amphiphile-loaded nanocarrier as a potentially therapeutic antibacterial against clinically relevant drug-resistant pathogenic bacteria.

Synthetic amphiphiles as therapeutic antibacterials: lessons on bactericidal efficacy and cytotoxicity and potential application as an adjuvant in antimicrobial chemotherapy.

In this paper, we present a critical assessment of the therapeutic potential of low molecular weight pyridine-based synthetic amphiphiles based on structure-guided bactericidal activity and a rational evaluation of their cytotoxic potential. Fluorescence-based structure-function studies revealed that the amphiphiles were membrane-acting and displayed a hierarchical pattern of bactericidal activity, which could be correlated with their charge density and hydrophobicity. The membrane-targeting activity of the most potent cationic amphiphile (compound 6) was vindicated as it induced extensive membrane-disruption and dissipation of the transmembrane potential (ΔΨ) in pathogenic bacteria. At concentrations equivalent to the minimum inhibitory concentration (MIC) against the Gram-positive pathogen S. aureus MTCC 96, none of the amphiphiles exerted any cytotoxic effect on model human cell lines (HeLa, MCF-7 and HT-29). However, at elevated concentrations, a distinct gradation in the cytotoxic effect was manifested, which is probably accounted by the charge density and conformational flexibility of the amphiphiles. A viable therapeutic application of compound 6 is demonstrated in combinatorial assays, wherein the proclivity of the amphiphile to disrupt bacterial membranes at very low concentration is exploited to enhance the uptake and bactericidal efficacy of erythromycin against Gram-negative pathogenic bacteria.