Modulating Helix-Preference of an Axially-Twisted Molecular Scaffold Through Diastereomeric Salt Formation with Tartaric Acid Stereoisomers.

Herein, we designed a chiral, axially-twisted molecular scaffold (ATMS) using pyridine-2,6-dicarboxamide (PDC) unit as pivot, chiral trans-cyclohexanediamine (CHDA) residues as linkers, and pyrene residues as fluorescent reporters. R,R-ATMS exclusively adopted M-helicity and produced differential response in UV-vis, fluorescence, and NMR upon addition of tartaric acid (TA) stereoisomers allowing naked-eye detection and enantiomeric content determination. Circular dichroism (CD) profile of R,R-ATMS underwent unique changes during titration with TA stereoisomers - while loss of CD signal at 345 nm was observed with equimolar D-TA and meso-TA, inversion was seen with equimolar L-TA. Temperature increase weakened these interactions to partially recover the original CD signature of R,R-ATMS. 2D NMR studies also indicated the significant structural changes in R,R-ATMS in the solution state upon addition of L-TA. Single crystal X-ray diffraction (SCXRD) studies on the crystals of the R,R-ATMS⊃D-TA salt revealed the interacting partners stacked in arrays and ATMS molecules stabilized by π-π stacking between its PDC and pyrene residues. Contrastingly, tightly-packed supramolecular cages comprised of four molecules each of R,R-ATMS and L-TA were seen in R,R-ATMS⊃L-TA salt, and the ATMS molecules contorted to achieve CH-π interactions between its pyrene residues. These results may have implications in modulating the helicity of topologically-similar larger biomolecules.

Divergent self-assembly propensity of enantiomeric phenylalanine amphiphiles that undergo pH-induced nanofiber-to-nanoglobule conversion.

This study presents the pathway diversity in the self-assembly of enantiomeric single phenylalanine derived amphiphiles (single F-PDAs), viz.L-NapF-EDA and D-NapF-EDA, that form supramolecular hydrogels at varied concentrations (≥1 mg mL-1 and ≥3 mg mL-1, respectively). By fitting the variable temperature circular dichroism (VT-CD) data to the isodesmic model, various thermodynamic parameters associated with their self-assembly, such as association constant (K), changes in enthalpy (ΔH), entropy (ΔS), and Gibbs free energy (ΔG), were extracted. The self-assembly of these single F-PDAs was found to be enthalpy-driven but entropically-disfavored. Although self-assembly of the D-isomer was slow, it also exhibited greater free energy of association than the L-isomer. Consequently, thermally and mechanically more robust self-assemblies were formed by the D-isomer than the L-isomer. We term these results as the "butterfly effect in self-assembly" wherein the difference in the stereochemical orientation of the residues at a single chiral center present in these molecules resulted in strong differences in the self-assembly propensity as well as in their thermal and mechanical stability. These single F-PDAs form helical nanofibers of opposite chirality upon self-assembly at basic pH (≥8) that produce intense CD signals. However, upon decreasing the pH, a gradual nanofiber-to-nanoglobular transformation was noticed due to protonation-induced structural changes in the PDAs.

Self-assembly of water-filled molecular saddles to generate diverse morphologies and high proton conductivity.

The design of single-component organic compounds acting as efficient solid-state proton conduction (SSPC) materials has been gaining significant traction in recent times. Molecular design and controlled self-assembly are critical components in achieving highly efficient SSPC. In this work, we report the design, synthesis, and self-assembly of an organic macrocyclic aza-crown-type compound, P2Mac, which complements synthetic ease with efficient SSPC. P2Mac is derived from the pyridine-2,6-dicarboxamide (PDC) framework and contains polar amide and amine residues in its inner region, while aromatic residues occupy the periphery of the macrocycle. The crystal structure analysis revealed that P2Mac adopts a saddle-shaped geometry. Each P2Mac molecule interacts with one water molecule that is present in its central polar cavity, stabilized by a network of five hydrogen bonds. We could self-assemble P2Mac in a variety of unique, aesthetically pleasing morphologies such as micron-sized octahedra, hexapods, as well as hollow nanoparticles, and microrods. The water-filled polar channels formed through the stacking of P2Mac allow attaining a high proton conductivity value of 21.1 mS cm-1 at 27 °C under a relative humidity (RH) of 95% in the single crystals of P2Mac, while the as-prepared P2Mac pellet sample exhibited about three-orders of magnitude lower conduction under these conditions. The low activation energy of 0.39 eV, calculated from the Arrhenius plot, indicates the presence of the Grotthus proton hopping mechanism in the transport process. This report highlights the pivotal role of molecular design and self-assembly in creating high-performance SSPC organic materials.

Exogenous application of nanocarrier-mediated double-stranded RNA manipulates physiological traits and defence response against bacterial diseases.

Stability and delivery are major challenges associated with exogenous double-stranded RNA (dsRNA) application into plants. We report the encapsulation and delivery of dsRNA in cationic poly-aspartic acid-derived polymer (CPP6) into plant cells. CPP6 stabilizes the dsRNAs during long exposure at varied temperatures and pH, and protects against RNase A degradation. CPP6 helps dsRNA uptake through roots or foliar spray and facilitates systemic movement to induce endogenous gene silencing. The fluorescence of Arabidopsis GFP-overexpressing transgenic plants was significantly reduced after infiltration with gfp-dsRNA-CPP6 by silencing of the transgene compared to plants treated only with gfp-dsRNA. The plant endogenous genes flowering locus T (FT) and phytochrome interacting factor 4 (PIF4) were downregulated by a foliar spray of ft-dsRNA-CPP6 and pif4-dsRNA-CPP6 in Arabidopsis, with delayed flowering and enhanced biomass. The rice PDS gene targeted by pds-dsRNA-CPP6 through root uptake was effectively silenced and plants showed a dwarf and albino phenotype. The NaCl-induced OsbZIP23 was targeted through root uptake of bzip23-dsRNA-CPP6 and showed reduced transcripts and seedling growth compared to treatment with naked dsRNA. The negative regulators of plant defence SDIR1 and SWEET14 were targeted through foliar spray to provide durable resistance against bacterial leaf blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo). Overall, the study demonstrates that transient silencing of plant endogenous genes using polymer-encapsulated dsRNA provides prolonged and durable resistance against Xoo, which could be a promising tool for crop protection and for sustaining productivity.

Underwater Adhesives from Redox-Responsive Polyplexes of Thiolated Polyamide Polyelectrolytes.

We report the fabrication of optically clear underwater adhesives using polyplexes of oppositely charged partially-thiolated polyamide polyelectrolytes (TPEs). The thiol content of the constituent PEs was varied to assess its influence on the adhesive properties of the resulting glues. These catechol-free, redox-responsive TPE-adhesives were formulated in aquo and exhibited high optical transparency and strong adhesion even on submerged or moist surfaces of diverse polar substrates such as glass, aluminium, wood, and bone pieces. The adhesives could be cured under water through oxidative disulphide crosslinking of the constituent TPEs. The polyamide backbone provided multi-site H-bonding interactions with the substrates while the disulphide crosslinking provided the cohesive strength to the glue. Strong adhesion of mammalian bones (load bearing capacity upto 7 kg/cm2 ) was achieved using the adhesive containing 30 mol % thiol residues. Higher pH and use of oxidants such as povidone-iodine solution enhanced the curing rate of the adhesives, and so did the use of Tris buffer instead of Phosphate buffer. The porous architecture of the adhesive and its progressive degradation in aqueous medium over the course of three weeks bode well for diverse biomedical applications where temporary adhesion of tissues is required.

Enzymatic Dimerization-Induced Self-Assembly of Alanine-Tyramine Conjugates into Versatile, Uniform, Enzyme-Loaded Organic Nanoparticles.

Herein, we report a novel enzymatic dimerization-induced self-assembly (e-DISA) procedure that converts alanine-tyramine conjugates into highly uniform enzyme-loaded nanoparticles (NPs) or nanocontainers by the action of horseradish peroxidase (HRP) in an aqueous medium under ambient conditions. The NP formation was possible with both enantiomers of alanine, and the average diameter could be varied from 150 nm to 250 nm (with a 5-12 % standard deviation of as-prepared samples) depending on the precursor concentration. About 60 % of the added HRP enzyme was entrapped within the NPs and was subsequently utilized for post-synthetic modification of the NPs with phenolic compounds such as tyramine or tannic acid. One-pot multi-enzyme entrapment of glucose oxidase (GOx) and peroxidase (HRP) within the NPs was also achieved. These GOx-HRP loaded NPs allowed multimodal detection of glucose, including that present in human saliva, with a limit of detection (LoD) of 740 nM through fluorimetry. The NPs exhibited good cytocompatibility and were stable to changes in pH (acidic to basic), temperature, ultrasonication, and even the presence of organic solvent (EtOH) to a certain extent, since they are stabilized by intermolecular hydrogen bonding, π-π, and CH-π interactions. The proposed e-DISA procedure can be widely expanded through the design of diverse enzyme-responsive precursors.

Molecular clips with spatially proximal urea residues for efficient transmembrane co-transport of H+/Cl- ions.

We report the design of bis(urea) functionalized amphiphilic molecular clips viz. 1a-1e to achieve efficient transmembrane co-transport of H+/Cl- ions. The most promising molecule 1a demonstrated a low nanomolar EC50 value (6.96 nM) to co-transport H+/Cl- ions via a carrier-mediated pathway and showed selective toxicity against cancerous HeLa cells as a result.

Luminescent hexagonal microtubes prepared through water-induced self-assembly of a polymorphic organoboron compound: formation mechanism and waveguide behaviour.

Tetra-coordinated organoboron (TCOB) compounds are promising candidates for developing high-performance optical devices due to their excellent optoelectronic performance. Fabricating TCOB-based nanomaterials of controlled and defined morphology through rapid and easy-to-execute protocols can significantly accelerate their practical utility in the aforesaid applications. Herein, we report water-induced self-assembly (WISA) to convert a polymorphic TCOB complex (HNBI-B, derived from a 2-(2'-hydroxy-naphthyl)-benzimidazole precursor) into two unique nanomorphologies viz. nanodiscoids (NDs) and fluorescent microtubes with hexagonal cross-sections (HMTs). Detailed electron microscopic investigations revealed that oriented assembly and fusion of the initially formed NDs yield the blue emissive HMTs (SSQY = 26.7%) that exhibited highly promising photophysical behaviour. For example, the HMTs outperformed all the crystal polymorphs of HNBI-B obtained from CHCl3, EtOAc and MeOH in emissivity and also exhibited superior waveguide behaviour, with a much lower optical loss coefficient α' = 1.692 dB mm-1 compared to the rod-shaped microcrystals of HNBI-B obtained from MeOH (α' = 1.853 dB mm-1). Thus, this work reports rapid access to high performance optical nanomaterials through WISA, opening new avenues for creating useful nanomaterial morphologies with superior optical performance.

Design of Cationic Lipids with Acetal Linkers: Conformational Preferences, Hydrolytic Stability, and High Drug-Loading Abilities.

Lipids are key constituents of numerous biomedical drug delivery technologies. Here, we present the design, synthesis and biophysical characterizations of a library of cationic lipids containing an acetal residue in their linker region. These cationic acetal lipids (CALs) were conveniently prepared through a trans-acetalization protocol from commercially available precursors. NMR studies highlighted the conformational rigidity at the acetal residue and the high hydrolytic stability of these CALs. Fluorescence anisotropy studies revealed that the CAL with a pyridinium headgroup (CAL1) formed highly cohesive vesicular aggregates in water. These structural and self-assembly features of the CAL1 allowed up to 196 % w/w loading of curcumin (Cur) as a representative hydrophobic drug. A reconstitutable formulation of Cur was obtained as a result, which could deliver the drug inside mammalian cells with very high efficiency. The hemocompatibility and cytocompatibility of CAL1 was significantly enhanced by creating a coating of polydopamine (PDA) onto its vesicular assemblies to produce hybrid lipid-polymer nanocapsules. This work demonstrates rapid access to the useful synthetic lipid formulations with high potential in drug and gene delivery applications.

A localized hydrogel-mediated chemotherapy causes immunogenic cell death via activation of ceramide-mediated unfolded protein response.

Treatment of triple-negative breast cancer (TNBC) is challenging because of its "COLD" tumor immunosuppressive microenvironment (TIME). Here, we present a hydrogel-mediated localized delivery of a combination of docetaxel (DTX) and carboplatin (CPT) (called DTX-CPT-Gel therapy) that ensured enhanced anticancer effect and tumor regression on multiple murine syngeneic and xenograft tumor models. DTX-CPT-Gel therapy modulated the TIME by an increase of antitumorigenic M1 macrophages, attenuation of myeloid-derived suppressor cells, and increase of granzyme B+CD8+ T cells. DTX-CPT-Gel therapy elevated ceramide levels in tumor tissues that activated the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)-mediated unfolded protein response (UPR). This UPR-mediated activation of apoptotic cell death led to release of damage-associated molecular patterns, thereby activating the immunogenic cell death that could even clear the metastatic tumors. This study provides a promising hydrogel-mediated platform for DTX-CPT therapy that induces tumor regression and effective immune modulation and, therefore, can be explored further for treatment of TNBC.

Bioinspired Design of an Uncharged Ambipolar Helical Scaffold To Achieve Efficient Solid-State Proton Conduction.

This work demonstrates highly efficient solid-state proton conduction in helical organic scaffolds inspired by the biomolecule gramicidin A. The scaffold, 1, derived from a pyridine-2,6-dicarboxamide (PDC) residue adopts a helical conformation that is stabilized by a network of strong bifurcated intramolecular H-bonds between the polar residues that align the inner (concave) face of the molecule, while the aromatic units in 1 are oriented outwards. As a result, the helix attains an ambipolar nature just like gramicidin A. Two different solid forms of 1 could be isolated: a yellow solid from high-polarity solvents and an orange solid from low-polarity solvents. Single-crystal X-ray diffraction (SCXRD) studies showed that in the former, molecules of 1 are stacked in a homochiral fashion, while in the latter heterochiral stacks of 1 were present. The yellow form exhibited an almost ∼300-fold higher conductivity (of up to 0.12 mS cm-1 at 95 °C and 95 % relative humidity) than the orange form as a result of closer intermolecular proximity and lower activation energy of 0.098 eV, thus indicating a Grotthus mechanism of proton transport. This study establishes the key role of bioinspired design and controlled stereo-organization of such discrete uncharged organic molecules in achieving efficient solid-state proton conduction.

Anion-responsive self-assembled hydrogels of a phenylalanine-TREN conjugate allow sequential release of propranolol and doxorubicin.

Stimuli-responsive self-assembled and supramolecular hydrogels derived from peptide amphiphiles have opened exciting new avenues in biomedicine and drug delivery. Herein, we screened a series of phenylalanine-amphiphiles possessing polyamine and oxyethylene appendages for their self-assembly and anion-responsiveness and found that the tris(aminoethyl)amine (TREN) containing amphiphile NapF-TREN formed injectable hydrogels that could be disrupted upon the addition of stoichiometric amounts of tetrahedral monovalent anions such as H2PO4- and HSO4-, while the addition of other anions such as Cl-, HPO42-, CO32-, HCO3- or SO42- did not affect the gel stability. The anion-gelator interaction was investigated by 1H and 31P NMR spectroscopy as well as by Isothermal Titration Calorimetry (ITC). These studies confirmed a 1 : 1 stoichiometry and revealed negative enthalpy and negative entropy for the binding of H2PO4- with NapF-TREN. Microscopic investigations by TEM, AFM, and SAXS revealed that H2PO4- anions induced a nanofiber-to-nanoglobule morphological change in the aqueous self-assemblies of NapF-TREN. However, upon ageing the samples, slow reformation of the nanofibers was also observed, reflecting the reversibility of the anion-gelator interaction. The anion- and pH-responsive nature of the NapF-TREN hydrogels was exploited to program sequential release of entrapped drugs propranolol and doxorubicin.

Hydrophobicity of Cholic Acid-Derived Amphiphiles Dictates the Antimicrobial Specificity.

The unique structural components of cell membranes of Gram-positive bacteria, Gram-negative bacteria, and mycobacteria provide an excellent therapeutic target for developing highly specific antimicrobials. Here, we report the synthesis of nine cholic acid (CA)-derived amphiphiles, where three hydroxyl groups of CA were tethered to dimethylamino pyridine and the C24-carboxyl group was conjugated with different alkyl chains. Structure-activity investigations revealed that amphiphile 1 harboring a methyl group has antimicrobial activity against mycobacterial species. On the other hand, amphiphile 7 containing an octyl chain was selective against Gram-positive and Gram-negative bacilli. Biochemical assays confirmed the selective membrane permeabilization abilities of amphiphiles 1 and 7. Importantly, we demonstrate the selective actions of amphiphiles in clearing biofilms, intracellular bacteria, and wound infections. Therefore, for the first time, we show that the unique structural features of CA-derived amphiphiles dictate selective activity against specific bacterial species.

Macrocyclic enforcement of twist in a secondary amide: reactivity and influence on photoisomerisation.

We report a penta-azamacrocycle (cDP2) containing a twisted secondary amide residue with a twist angle (τ) of 28° and pyramidalization parameter (χN) of 53°. Consequently, cDP2 is reactive towards a host of nucleophiles such as amines, thiols and hydroxyls. Moreover, the twisted amide residue also facilitates the photo-isomerisation of the diazobenzene residue of cDP2.

Polyaspartate-derived synthetic antimicrobial polymer enhances the activity of rifampicin against multidrug-resistant Pseudomonas aeruginosa infections.

Infections caused by multidrug-resistant Pseudomonas aeruginosa (P. aeruginosa) pose major challenges for treatment due to the acquired, adaptive, and intrinsic resistance developed by the bacteria. Accumulation of mutations, the ability to form biofilms, and the presence of lipopolysaccharides in the outer bacterial membranes are the key mechanisms of drug resistance. Here, we show that a polyaspartate-derived synthetic antimicrobial polymer (SAMP) with a hexyl chain (TAC6) is an effective adjuvant for a hydrophobic antibiotic, rifampicin. Our in vitro studies demonstrated that the combination of TAC6 and rifampicin is effective against clinically isolated multidrug-resistant strains of P. aeruginosa. Membrane permeabilization studies showed that TAC6 allows the permeabilization of bacterial membranes, and the accumulation of rifampicin inside the cells, thereby enhancing its activity. The combination of TAC6 and rifampicin can also degrade the P. aeruginosa biofilms, and therefore can mitigate the adaptive resistance developed by bacteria. We further demonstrated that the combination of TAC6 and rifampicin can clear P. aeruginosa-mediated wound infections effectively. Therefore, our study showed polyaspartate-derived SAMP to be an effective antibiotic adjuvant against P. aeruginosa infections.

Hydrogel-mediated topical delivery of steroids can effectively alleviate psoriasis via attenuating the autoimmune responses.

Psoriasis is a systemic, relapsing, and chronic autoimmune inflammatory disease of the skin. Topical use of betamethasone, a glucocorticoid, in the form of creams is a common treatment for psoriasis. However, topical use of these creams is challenging due to the ineffective entrapment of steroids, burst release of the entrapped drugs, poor skin permeability, and high toxicity. Herein, we present the engineering of a betamethasone-loaded topical hydrogel (B-Gel) that can efficiently entrap steroids with high spreadability, and can also maintain the sustained release of drugs. We used an imiquimod (IMQ) induced ear psoriasis model, and demonstrated that topical application of B-Gel can mitigate the autoimmune inflammation reactions, and leads to a reduction in erythema, induration, scaling, and ear thickness. As interleukin 17 (IL-17) secreting T helper 17 (Th17) cells and γδ+ T cells are responsible for psoriasis, B-Gel treatment witnessed a reduction in the infiltration of leukocytes, CD4+ T cells, Th17 T cells, and dermal γδ+ T cells. We further demonstrated that B-Gel mediated reduction of IL-1ß, IL-17, and K16 (marker for keratinocyte proliferation) is responsible for alleviation of psoriasis. Therefore, the non-greasy nature of the hydrogel with a cooling effect provides an alternative for topical application of steroids.

Nonimmunogenic Hydrogel-Mediated Delivery of Antibiotics Outperforms Clinically Used Formulations in Mitigating Wound Infections.

Treatment of chronic wound infections caused by Gram-positive bacteria such as Staphylococcus aureus is highly challenging due to the low efficacy of existing formulations, thereby leading to drug resistance. Herein, we present the synthesis of a nonimmunogenic cholic acid-glycine-glycine conjugate (A6) that self-assembles into a supramolecular viscoelastic hydrogel (A6 gel) suitable for topical applications. The A6 hydrogel can entrap different antibiotics with high efficacy without compromising its viscoelastic behavior. Activities against different bacterial species using a disc diffusion assay demonstrated the antimicrobial effect of the ciprofloxacin-loaded A6 hydrogel (CPF-Gel). Immune profiling and gene expression studies after the application of the A6 gel to mice confirmed its nonimmunogenic nature to host tissues. We further demonstrated that topical application of CPF-Gel clears S. aureus-mediated wound infections more effectively than clinically used formulations. Therefore, cholic acid-derived hydrogels are an efficacious matrix for topical delivery of antibiotics and should be explored further.

A hydrogel-based implantable multidrug antitubercular formulation outperforms oral delivery.

We present a non-immunogenic, injectable, low molecular weight, amphiphilic hydrogel-based drug delivery system (TB-Gel) that can entrap a cocktail of four front-line antitubercular drugs, isoniazid, rifampicin, pyrazinamide, and ethambutol. We showed that TB-Gel is more effective than oral delivery of the combination of four drugs in reducing the mycobacterial infection in mice. Results show that half the dose of chemotherapeutic drugs is sufficient to achieve a comparable therapeutic effect to that of oral delivery.

Polydopamine and dopamine interfere with tetrazolium-based cytotoxicity assays and produce exaggerated cytocompatibility inferences.

Tetrazolium-based assays such as the MTT assay have been commonly employed in evaluating biocompatibility. Here, we show that PDA (or its precursor dopamine (DA)) spontaneously reduces MTT and produces exaggerated cytocompatibility inferences. The extent of interference depends on the method of DA polymerization. We observed that the trypan blue exclusion assay allowed more accurate determination of cell viability in the presence of DA- and PDA-based nanomaterials.

Nanoparticle-mediated gene therapy strategies for mitigating inflammatory bowel disease.

Inflammatory bowel disease (IBD) is an autoimmune disorder of the gastrointestinal tract (GIT) where Ulcerative Colitis (UC) displays localized inflammation in the colon, and Crohn's Disease (CD) affects the entire GIT. Failure of current therapies and associated side-effects bring forth serious social, economic, and health challenges. The gut epithelium provides the best target for gene therapy delivery vehicles to combat IBD. Gene therapy involving the use of nucleic acid (NA) therapeutics faces major challenges due to the hydrophilic, negative-charge, and degradable nature of NAs. Recent success in the engineering of biomaterials for gene therapy and their emergence in clinical trials for various diseases is an inspiration for scientists to develop gene therapy vehicles that can be easily targeted to the desired tissues for IBD. Advances in nanotechnology have enabled the formulations of numerous nanoparticles for NA delivery to mitigate IBD that still faces challenges of stability in the GIT, poor therapeutic efficacy, and targetability. This review presents the challenges of gene therapeutics, gastrointestinal barriers, and recent advances in the engineering of nanoparticles for IBD treatment along with future directions for successful translation of nanoparticle-mediated gene therapeutics in clinics.