Cumulative Effect of pH and Redox Triggers on Highly Adaptive Transient Coacervates.

An intricate synergism between multiple biochemical processes and physical conditions determines the formation and function of various biological self-assemblies. Thus, a complex set of variables dictate the far-from-equilibrium nature of these biological assemblies. Mimicking such systems synthetically is a challenging task. We report multi-stimuli responsive transient coacervation of an aldehyde-appended polymer and a short peptide. The coacervates are formed by the disulphide linkages between the peptide molecules and the imine bond between the polymer and the peptide. Imines are susceptible to pH changes and the disulphide bonds can be tuned by oxidation/reduction processes. Thus, the coacervation is operational only under the combined effect of appropriate pH and oxidative conditions. Taking advantage of these facts, the coacervates are transiently formed under a pH cycle (urea-urease/gluconolactone) and a non-equilibrium redox cycle (TCEP/H2 O2 ). Importantly, the system showed high adaptability toward environmental changes. The transient existence of the coacervates can be generated without any apparent change in size and shape within the same system through the sequential application of the above-mentioned nonequilibrium reaction cycles. Additionally, the coacervation allows for efficient encapsulation/stabilisation of proteins. Thus, the system has the potential to be used for protein/drug delivery purposes in the future.

Mannose-Decorated Composite Peptide Hydrogel with Thixotropic and Syneresis Properties and its Application in Treatment of Leishmaniasis.

Leishmaniasis, caused by the intramacrophage protozoan parasite Leishmania donovani, is a life-threatening yet neglected vector-borne disease. Few medications for the treatment of this disease are available. However, targeted delivery of drugs to macrophages remains a significant concern. Macrophages are equipped with many receptors, and therefore putting suitable ligands in the macrophage targeting drug delivery vehicle gained a lot of attention. One such receptor is the mannose receptor, abundantly expressed by macrophages. To treat this deadly disease, in this study, a mannose containing composite hydrogel is prepared by combining a self-aggregating short peptide (Nap-FFGE-NH2 , Pep-A) and a mannose containing non-aggregating peptide (Nap-FF-mannosyl, Pep-B). The self-aggregation of the composite hydrogel is evaluated using various spectroscopic and microscopic techniques. Intermolecular hydrogen bonding and π-π stacking lead to an antiparallel ß-sheet like arrangement of the peptides. Notably, the composite hydrogel showed shear-thinning and syneresis properties. Moreover, the composite hydrogel was found to be stable in cell-culture media, biodegradable and non-toxic to the macrophages. Both control and infected macrophages showed effective cell growth and proliferation when subjected to the composite 2D and 3D hydrogel matrix. When treated with Amphotericin B loaded composite hydrogel, the drug was effectively delivered to kill the parasite in the infected macrophages. Almost 3.5 fold decrease in the parasite burden was recorded when infected cells were treated with drug-loaded composite hydrogel. The injectability, biodegradability, non-cytotoxicity, and efficient drug delivery properties of the mannose-functionalized hydrogel make it a suitable candidate for the treatment of Leishmaniasis.

Crosslinker-free collagen gelation for corneal regeneration.

Development of an artificial cornea can potentially fulfil the demand of donor corneas for transplantation as the number of donors is far less than needed to treat corneal blindness. Collagen-based artificial corneas stand out as a regenerative option, having promising clinical outcomes. Collagen crosslinked with chemical crosslinkers which modify the parent functional groups of collagen. However, crosslinkers are usually cytotoxic, so crosslinkers need to be removed from implants completely before application in humans. In addition, crosslinked products are mechanically weak and susceptible to enzymatic degradation. We developed a crosslinker free supramolecular gelation strategy using pyrene conjugated dipeptide amphiphile (PyKC) consisting of lysine and cysteine; in which collagen molecules are intertwined inside the PyKC network without any functional group modification of the collagen. The newly developed collagen implants (Coll-PyKC) are optically transparent and can effectively block UV light, are mechanically and enzymatically stable, and can be sutured. The Coll-PyKC implants support the growth and function of all corneal cells, trigger anti-inflammatory differentiation while suppressing the pro-inflammatory differentiation of human monocytes. Coll-PyKC implants can restrict human adenovirus propagation. Therefore, this crosslinker-free strategy can be used for the repair, healing, and regeneration of the cornea, and potentially other damaged organs of the body.

Development of a hydrolase mimicking peptide amphiphile and its immobilization on silica surface for stereoselective and enhanced catalysis.

Biocatalysis is an important area of modern research and is extensively explored by various industries to attain greener methods in various applications. Supramolecular interactions of short peptides have been under the scanner for developing artificial smart materials inspired from natural systems. Peptide-based artificial enzymes have been proved to show various enzyme-like activities. Therefore, immobilization of catalytic peptides on solid surfaces can be an extremely useful breakthrough for development of cost-effective catalytic formulations. In this work, a series of peptide amphiphiles (PAs) have been systematically analyzed to find the most effective catalyst with esterase like activity. The PA, containing a catalytic triad, 'Asp(Ser)His' in a branched manner, was further immobilized onto silica nanoparticles through covalent bonding method to obtain surface coated catalytic silica nanoparticles. The heterogenous catalytic formulation not only showed enhanced esterase activity than the self-assembled PA in homogenous phase, but also exceeded the activity of natural CV lipase. The catalytic formulation showed high stereoselectivity towards chiral esters. Moreover, the catalyst remained stable at higher temperature, in presence of various denaturant and retained its activity after several catalytic cycles. The ease of separation, robust nature, reusability and high stereoselectivity of the catalyst opens up the possibility of creating new generation heterogeneous catalysts for further industrial applications.

The Effects of a Short Self-Assembling Peptide on the Physical and Biological Properties of Biopolymer Hydrogels.

Hydrogel scaffolds have attracted much interest in the last few years for applications in the field of bone and cartilage tissue engineering. These scaffolds serve as a convenient three-dimensional structure on which cells can grow while sensing the native environment. Natural polymer-based hydrogels are an interesting choice for such purposes, but they lack the required mechanical properties. In contrast, composite hydrogels formed by biopolymers and short peptide hydrogelators possess mechanical characteristics suitable for osteogenesis. Here, we describe how combining the short peptide hydrogelator, Pyrene-Lysine-Cysteine (PyKC), with other biopolymers, can produce materials that are suitable for tissue engineering purposes. The presence of PyKC considerably enhances the strength and water content of the composite hydrogels, and confers thixotropic behavior. The hyaluronic acid-PyKC composite hydrogels were shown to be biocompatible, with the ability to support osteogenesis, since MC3 T3-E1 osteoblast progenitor cells grown on the materials displayed matrix calcification and osteogenic differentiation. The osteogenesis results and the injectability of these composite hydrogels hold promise for their future utilization in tissue engineering.

Smart Thixotropic Hydrogels by Disulfide-Linked Short Peptides for Effective Three-Dimensional Cell Proliferation.

Supramolecular assembly of short peptides is a crucial process and has shown numerous potential applications as biomaterials. In the present work, the hydrogelation process of short peptides containing C-terminal "Lys-Cys" (KC) residues have been studied in detail. The N-terminal capping is found to be essential for effective gelation. Out of 12 peptides we studied, two of them could form hydrogels efficiently: Ac-VVKC-NH2 and Ac-FFKC-NH2. In both cases, the monomer-to-dimer formation through disulfide linkages by Cys residues controls the aggregation process. Interestingly, the presence of H2O2 facilitated the dimerization and thereby reduced the gelation time but could not impart much effect on the mechanical properties of the gels. Detailed rheological study revealed that both hydrogels are thixotropic in nature. Moreover, they are responsive to glutathione (GSH) due to the presence of disulfide linkages. However, the hydrogel of Ac-FFKC-NH2 is found to be stronger and more effective for biological applications. The thixotropic nature as well as a model drug release study in response to varying GSH concentration indicates the possible use of the hydrogel as an injectable local drug delivery vehicle. The hydrogel of Ac-FFKC-NH2 is noncytotoxic in nature. Three-dimensional cell proliferation has been found to be more effective than 2D, as it mimics the in vivo situation more closely if not exactly. In the present study, we have shown that both differentiated RAW macrophages and undifferentiated THP-1 monocytes could proliferate significantly within the 3D matrix of the hydrogel, without depicting any apparent cytotoxicity. Thus, the hydrogel of Ac-FFKC-NH2 has potential for application in localized drug administration and as a supporting biomaterial to study basic phenomena involving cell behavior.

Light-triggered syneresis of a water insoluble peptide-hydrogel effectively removes small molecule waste contaminants.

A short peptide based hydrogel exhibits aqueous insolubility, thixotropy and efficient light induced syneresis. Upon irradiation with UV light, the hydrogel shrinks and expells ∼50% of the solvent. Syneresis is caused by light-triggered trans-cis isomerisation of an azobenzene moiety in the peptide derivative. This expulsion of solvent can be effectively exploited in the removal of low molecular weight contaminants in water.

Peptide Hydrogels as Platforms for Sustained Release of Antimicrobial and Antitumor Drugs and Proteins.

A charged synthetic peptide-based noncytotoxic hydrogelator was employed in encapsulation, storage, and sustainable release of different kinds of drugs, namely, ciprofloxacin (CP), an antibiotic; 5-fluorouracil (5-FU), an anticancer drug and proteins like lysozyme and bovine serum albumin (BSA). Hydrogelation of the peptide and its coassembly with the drug molecules were studied to obtain mechanistic details. All of the different cargos were capable of sustained and efficient release from the delivery platform. The drugs were found to retain their activity post release, while the proteins showed complete retention of their secondary structure. While about 80% CP was released at physiological pH over a period of 3 days, 5-FU was better released (73%) at an acidic pH (5.5) in comparison to the physiological pH (68%). Lysozyme was better released (82%) than BSA (43%) owing to the smaller size of the former and negative charge on the latter. Such biocompatible multicargo-releasing platforms from simple economically viable biomaterials, capable of sustained and tissue-specific release of cargo, are extremely promising in topical delivery of therapeutics.

Unusual confinement properties of a water insoluble small peptide hydrogel.

Unlike polymeric hydrogels, in the case of supramolecular hydrogels, the cross-linked network formation is governed by non-covalent forces. Hence, in these cases, the gelator molecules inside the network retain their characteristic physicochemical properties as no covalent modification is involved. Supramolecular hydrogels thus get dissolved easily in aqueous medium as the dissolution leads to a gain in entropy. Thus, any supramolecular hydrogel, insoluble in bulk water, is beyond the present understanding and hitherto not reported as well. Herein, we present a peptide-based (PyKC) hydrogel which remained insoluble in water for more than a year. Moreover, in the gel state, any movement of solvent or solute to and from the hydrogel is highly restricted resulting in a high degree of compartmentalization. The hydrogel could be re-dissolved in the presence of some biomolecules which makes it a prospective material for in vivo applications. Experimental studies and all atom molecular dynamics simulations revealed that a cysteine containing gelator forms dimers through disulfide linkage which self-assemble into PyKC layers with a distinct PyKC-water interface. The hydrogel is stabilized by intra-molecular hydrogen bonds within the peptide-conjugates and the π-π stacking of the pyrene rings. The unique confinement ability of the hydrogel is attributed to the slow dynamics of water which remains confined in the core region of PyKC via hydrogen bonds. The hydrogen bonds present in the confined water need activation energies to move through the water depleted hydrophobic environment of pyrene rings which significantly reduces water transport across the hydrogel. The compartmentalizing ability is effectively used to protect enzymes for a long time from denaturing agents like urea, heat or methanol. Overall, the presented system shows unique insolubility and confinement properties that could be a milestone in the research of soft-materials.

Bis-arylidene oxindole-betulinic Acid conjugate: a fluorescent cancer cell detector with potent anticancer activity.

Molecules offering simultaneous detection and killing of cancer cells are advantageous. Hybrid of cancer cell-selective, ROS generator betulinic acid and bis-arylidene oxindole with amino propyl-linker is developed. With intrinsic fluorescence, the molecule exhibited cancer cell-specific residence. Further, it generated ROS, triggered apoptosis, and exhibited potent cytotoxicity in cancer cells selectively. We demonstrate the first example and use of isatins as betulinic acid conjugate for selective detection of cancer and subsequent killing of cancer cells via apoptosis.