Vignesh Muthuvijayan

A novel method of preparing chitosan gels using in situ generated negatively-charged colloidal salts of a variety of metal ions is described. Their potential as scaffolds for tissue-engineering and as recoverable catalysts in aza-Michael addition is demonstrated here. Given their wide range of properties, they have broad scope for applications.

Biomimicry is becoming deep-rooted as part of bioceramics owing to its numerous functional advantages. Naturally occurring hydroxyapatite (HA) apart from primary nano structures are also characterised by various ionic substitutions. The ease of accommodating such key elements into the HA lattice is known to enhance bone healing properties of bioceramics. In this work, hydroxyapatite synthesized via biomimetic approach was substituted with individual as well as multiple cations for potential applications in bone repair. Ion substitutions of Sr, Mg and Zn was carried out on HA for the first time by using Serratia grown in a defined biomineralization medium. The individual ions of varying concentration substituted in Serratia HA (SHA) (Sr SHA, Mg SHA and Zn SHA) were analysed for crystallinity, functional groups, morphology and crystal size. All three showed decreased crystallinity, phase purity, large agglomerated aggregates and needle-shaped morphologies. Fourier transform infrared spectroscopy (FTIR) spectra indicated increased carbonate content of 5.8% resembling that of natural bone. Additionally, the reduced O-H intensities clearly portrayed disruption of HA lattice and subsequent ion-substitution. The novelty of this study lies primarily in investigating the co-substitution of a combination of 1% Sr, Zn and Mg in SHA and establishing the associated change in bone parameters. Scanning electron microscope (SEM) and transmission electron microscope (TEM) images clearly illustrated uniform nano-sized agglomerates of average dimensions of 20-50 nm length and 8-15 nm width for Sr SHA; 10-40 nm length and 8-10 nm width for both Zn SHA and Mg SHA and 40-70 nm length and 4-10 nm width in the case of 1% Sr, Zn, Mg SHA. In both individual as well as co-substitutions, significant peak shifts were not observed possibly due to the lower concentrations. However, cell volumes increased in both cases due to presence of Sr2+ validating its dominant integration into the SHA lattice. Rich trace ion deposition was presented by energy dispersive X-ray spectroscopy (EDS) and quantified using inductively coupled plasma optical emission spectrometer (ICP-OES). In vitro cytotoxicity studies in three cell lines viz. NIH/3T3 fibroblast cells, MG-63 osteosarcoma cells and RAW 264.7 macrophages showed more than 90% cell viability proving the biocompatible nature of 1% Sr, Zn and Mg in SHA. Microbial biomineralization by Serratia produced nanocrystals of HA that mimicked "bone-like apatite" as evidenced by pure phase, carbonated groups, reduced crystallinity, nano agglomerates, variations in cell parameters, rich ion deposition and non-toxic nature. Therefore ion-substituted and co-substituted biomineralized nano SHA appears to be a suitable candidate for applications in biomedicine addressing bone injuries and aiding regeneration as a result of its characteristics close to that of the human bone.

The authors regret that the following equation, which was used for calculating cell viability in section 2.5.1.1, was inadvertently missed in the final version of the manuscript during proofreading. [Formula presented] The authors would like to apologize for any inconvenience caused.

Poor haemocompatibility of material surfaces is a serious limitation that can lead to failure of blood-contacting devices and implants. In this work, we have improved the haemocompatibility of polyethylene terephthalate (PET) surfaces by immobilizing apyrase/ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) on to the carboxylated PET. NTPDase immobilized PET surfaces scavenge the ADP released by activated platelets, which prevents further platelet activation and aggregation. The surface properties of the modified PET were characterized by scanning electron microscope (SEM), energy dispersive X-ray spectroscopy (EDAX), and contact angle measurement. The enzyme attachment and stability on the modified PET surfaces were evaluated. The kinetics of free enzyme and immobilized enzyme were studied and fitted using the Michaelis-Menten kinetic model. Both free and immobilized NTPDase followed Michaelis-Menten kinetics with similar Michaelis-Menten constants (K m ). This suggests that the activity of NTPDase was unchanged upon immobilization. Protein adsorption and %hemolysis was significantly reduced for carboxylated PET and NTPDase immobilized PET surfaces compared to unmodified PET. Lactate dehydrogenase assay showed that the number of adhered platelets reduced by more than an order of magnitude for the NTPDase immobilized PET surface compared to unmodified PET. These results clearly indicate that NTPDase immobilization significantly enhances the haemocompatibility of PET surfaces.

In this research, biomimetic deposition of “bone-like” apatite by novel Gram-positive bacterium Bacillus tequilensis was investigated. Hydroxyapatite (HA) was produced by Bacillus tequilensis using defined biomineralization media and dried. Calcination was carried out at different temperatures (100 °C–900 °C) and HA nanopowder was analysed for its structural phase composition, crystallinity, crystallite size and functional groups. X-ray diffractometry (XRD) indicated that increasing temperatures increased the crystallinity of HA nanocrystals. The presence of carbonate groups was evidenced by Fourier transform infrared (FTIR) spectrum and the purity of synthesized apatite nanocrystals was validated by absence of secondary peaks in XRD studies. Scanning electron microscopy (SEM) images depicted those uniform spherical agglomerates of HA comprised of nanosized crystallites. Transmission electron microscope (TEM) results identified needle-like crystal morphologies with average dimensions of 30–60 nm length and 3–10 nm width. Rich trace ion deposition was illustrated by energy dispersive x-ray spectroscopy (EDS) and quantified using inductively coupled plasma optical emission spectrometer (ICP-OES). Overall, microbial biomineralization by Bacillus tequilensis produced nanocrystals of HA that mimicked “bone-like apatite” as evidenced by pure phase, B-type carbonated form, poor crystallinity and trace amounts of vital elements (Mg, Na, K, Zn, Sr, Cl). Moreover, in vitro cytotoxicity studies revealed more than 80% cell viability highlighting the biocompatible nature of synthesized nano HA. Thereby, Bacillus tequilensis biomineralized nano HA reflects as a suitable candidate for applications in biomedicine addressing bone injuries and aiding regeneration.

Fluorophores are used for sensing biologically relevant ions, toxic metals or pathogenic markers. However, the mode of entry of such fluorophores into the cell greatly depends on their size, shape, surface charge, functional groups, and hydrophobicity. In particular, the influence of hydrophobicity on the intracellular uptake of fluorophores is poorly investigated. Self-assembly is a recent strategy to tune the intracellular uptake of fluorophores, facilitating increased intracellular sensing and fluorescence. Herein, self-assembly of three novel poly(aryl ether) dendron derivatives that contain rhodamine units was used to investigate the effect of hydrophobicity on the intracellular uptake of self-assembled fluorophores. The results suggest that monomer hydrophobicity plays an important role in the uptake. The dendron-based fluorophores, which upon self-assembly, formed stable spherical aggregates ranging from 300 to 500 nm. The rhodamine-based dendrons could selectively sense Hg 2+ ions in the presence of other competing metal cations. Intracellular imaging of the dendron-based fluorophores displayed bright red fluorescence in human embryonic kidney cells. The rate of intracellular uptake of the three dendron-based fluorophores was analyzed by flow cytometry. The results establish the importance of the hydrophilic-lipophilic balance of the self-assembled amphiphiles for tuning the intracellular uptake.

The increased threat of bacterial resistance against conventional antibiotics has warranted the need for development of membrane targeting antibacterial agents. Several self-assembled cationic amphiphiles with different supramolecular structures have been reported in recent years for potent antibacterial activity with increased specificity. In this study, we describe the self-assembly and antibacterial activity of four lower generation poly(aryl ether)-based amphiphilic dendrimers (AD-1, AD-2, AD-3, and AD-4) containing terminal amine (PAMAM-based), ester, and hydrazide functional groups with varied hydrophobicity. Among the four dendrimers under study, the amine-terminated dendrimer (AD-1) displayed potent antibacterial activity. The ratio of surface cationic charge to hydrophobicity had a significant effect on the antibacterial activity, where AD-3 dendrimer with increased surface cationic charges exhibited a higher minimum inhibitory concentration (MIC) than AD-1. AD-2 (ester terminated) and AD-4 (hydrazide terminated) dendrimers did not show any bactericidal activity. The amphiphilic dendrimer-bacteria interactions, further validated by binding studies, also showed significant changes in bacterial morphology, effective membrane permeation, and depolarization by AD-1 in comparison with AD-3. Molecular dynamics simulations of AD-1 and AD-3 on bacterial membrane patches further corroborated the experimental findings. The structural conformation of AD-1 dendrimer facilitated increased membrane interaction compared to AD-3 dendrimer. AD-1 also displayed selectivity to bacterial membranes over fibroblast cells (4× MIC), corroborating the significance of an optimal hydrophobicity for potent antibacterial activity with no cytotoxicity. The self-assembled (poly(aryl ether)-PAMAM-based) dendrimer (AD-1) also exhibited potent antibacterial activity in comparison with conventional higher generation dendrimers, establishing the implication of self-assembly for bactericidal activity. Moreover, the detailed mechanistic study reveals that optimal tuning of the hydrophobicity of amphiphilic dendrimers plays a crucial role in membrane disruption of bacteria. We believe that this study will provide valuable insights into the design strategies of amphiphilic dendrimers as antibacterial agents for efficient membrane disruption.

In this study, a pH-induced self-assembly-based method has been developed to form silk fibroin nanoparticles (SFN-2) with a higher drug loading capacity (21.0 ± 2.1%) and cellular uptake than that of silk fibroin particles produced by a conventional desolvation method (SFN-1). Using the self-assembly method, rifampicin-encapsulated silk fibroin nanoparticles (R-SFN-2) were prepared with a size of 165 ± 38 nm at an optimum pH of 3.8. In silico analysis reveals that at acidic pH, the amino acid side chain charge neutralization of acidic residues, especially GLU64, promotes the formation of additional favorable interactions between the silk fibroin and the drug. The SFN-2 also possess a good aerosol property with a mass median aerodynamic diameter of 3.82 ± 0.71 μm and fine particle fraction of 64.0 ± 1.4%. These SFN-2 particles were selectively endocytosed by macrophages through clathrin- and caveolae-mediated endocytosis with a higher uptake efficiency (66.2 ± 2.1%) and were found to exhibit a sustained drug release in the presence of macrophage intracellular lysates. The cytokine and biomarker expression analyses revealed that SFN-2 could exhibit an immunomodulatory effect by polarizing the macrophages to an initial M1 phase and later M2 phase. Further, R-SFN-2 also exhibited an enhanced and sustained intracellular antibacterial activity against Mycobacterium smegmatis-infected macrophages than free rifampicin. Thus, the self-assembled silk fibroin particles with immunomodulatory action combined with a good aerosol and intracellular drug release property can be an attractive choice as a carrier for developing pulmonary drug delivery systems.

Supramolecular micelles are of particular interest in cancer therapy, owing to their capability to enable the on-demand drug release in tumor microenvironments. Nano platforms of supramolecular micelles can be facilely achieved from β-cyclodextrin (β-CD) based polymers. Herein, we developed novel supramolecular micelles of β-CD-(PNVP)4-AD-PLGA produced from β-CD-grafted star poly(N-vinylpyrrolidone) (β-CD-(PNVP)4) and adamantine-terminated linear-poly(lactic-co-glycolide) (AD-PLGA) by host-guest interaction between β-cyclodextrin and adamantine groups. A model anticancer drug, doxorubicin (DOX) was loaded into the supramolecular micelles during self-assembly in aqueous solution. The dissociation of the supramolecular micelles was triggered by acidic environments, which led to the release of DOX more in pH 6.4 compared to pH 7.4. The in vitro toxicity of the drug loaded micelles revealed 87% of cytotoxicity after 72 h against glioblastoma (C6 cells) while β-CD-(PNVP)4-AD-PLGA was biocompatible to HEK 293 cells (non-cancerous). Furthermore, β-CD-(PNVP)4-AD-PLGA and β-CD-(PNVP)4-AD-PLGA/DOX were found to be hemocompatible and more suitable for Intravenous administration. Our results suggest that the developed micelle system can provide a promising robust and sustained anticancer drug delivery system for the future cancer treatment.