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.

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.

Bi-directional feedback controlled transience in Cucurbituril based tandem nanozyme.

Life is fueled by multi-enzymatic tandem processes that display unmatched catalytic efficiencies owing to certain features of the biological reactors such as compartmentalization, nano-confinement, and out-of-equilibrium dynamics. With an attempt to match such natural catalytic systems, herein, we present a chemoenzymatic pH clock mediated transient assembly of a vesicular nanozyme. Distinct confinement of two catalytically discrete units, Histidine groups on the periphery and hemin in the lipid bilayer, results in an efficient hydrolase-peroxidase tandem catalysis in a temporally controlled fashion. The pH clock, constituted by alkaline TRIS (Tris(hydroxymethyl)aminomethane hydrochloride) buffer (promoter) and glucose oxidase (GOx) catalyzed oxidation of glucose, steers the transience in an asymmetric fashion. Alkaline TRIS buffer enhances the pH of the system and triggers the formation of imine linked Supramolecular Peptide Amphiphiles (SPAs) that further assemble into vesicles. On the other hand, oxidation of glucose produces gluconolactone and H2O2. Gluconolactone hydrolyzes to gluconic acid (deactivator) which dissipates the nanozyme while H2O2 is used in the peroxidase catalysis. Thus, the bi-directional feedback from the fuel not only regulates the existence of the transient state but also controls the activity of the assembly. The transiently assembled nanozyme protected the activity of the catalytic units, displayed substrate specificity and catalytic reproducibility over multiple fueling cycles.

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.

pH clock instructed transient supramolecular peptide amphiphile and its vesicular assembly.

A new strategy to construct a transient supramolecular peptide amphiphile (SPA) and its vesicular aggregates is displayed. The construction of the amphiphile is assisted by the ternary complexation of cucurbit[8]uril and pH responsive imine bond formation. The transient assembly follows a pH clock set by urea/urease and hydrolysis of glucono delta-lactone (GdL). The transient assembly can be repeated for several cycles through feeding the system with the fuel (urea).

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.

Unorthodox Combination of Cation-π and Charge-Transfer Interactions within a Donor-Acceptor Pair.

Cation-π and charge-transfer (CT) interactions are ubiquitous in nature and involved in several biological processes. Although the origin of both the interactions in isolated pairs has extensively been studied, CT interactions are more prominent in supramolecular chemistry. Involvement of cation-π interactions in the preparation of advanced functional soft materials is uncommon. Moreover, a combination of these two interactions within a pair of electron donor (D) and acceptor (A) is uncharted. Here, we present a rational design to incorporate a combination of these two interactions within a D-A pair. A pyrene-peptide conjugate exhibits a combination of cation-π and CT interactions with a cationic naphthalenediimide (NDI) molecule in water. Nuclear Overhauser effect spectroscopy NMR along with other techniques and density functional theory calculations reveal the involvement of these interactions. The π-planes of pyrene and NDI adopt an angle of 56° to satisfy both the interactions, whereas ß-sheet formation by the peptide sequence facilitates self-assembly. Notably, the binary system forms a self-supporting hydrogel at a higher concentration. The hydrogel shows efficient self-healing and injectable property. The hydrogel retains its thixotropic nature even at an elevated temperature. Broadly, we demonstrate a pathway that should prove pertinent to various areas, ranging from understanding biological assembly to peptide-based functional soft materials.

Multiple Cross-Linking of a Small Peptide to Form a Size Tunable Biopolymer with Efficient Cell Adhesion and Proliferation Property.

Development of biocompatible polymeric systems capable of cell adhesion and proliferation is a challenging task. Proper cross-linking of small cell adhesive peptide sequences is useful in this respect as it provides the inherent nontoxic environment as well as the cross-linked polymeric network to the cells for adhesion and proliferation. A multiple cross-linking strategy is applied to create a peptide-based cross-linked polymer. Covalent linkage through disulfide bond formation, supramolecular linkage using homoternary complexation by CB[8], and enzymatic cross-linking by HRP-mediated dimerization of tyrosine are used to prepare the cross-linked, peptide-based polymer decorated with cell-adhesive RGDS sequence. The supramolecular cross-linking via CB[8] provided stability as well as brings the RGDS sequences at the surface of the polymer particles. The order of cross-linking allowed to fine-tune the particle size of the polymer and polymer particles of wide range (200-1000 nm) can be prepared by varying the order. The cross-linked polymer particles (P1 and P2) were found to be stable at wide range of temperature and pH. Moreover, as intended, the polymer was noncytotoxic in nature and showed efficient cell adhesion and proliferation property, which can be used for further biological applications.

Solvent Assisted Tuning of Morphology of a Peptide-Perylenediimide Conjugate: Helical Fibers to Nano-Rings and their Differential Semiconductivity.

Understanding the regulatory factors of self-assembly processes is a necessity in order to modulate the nano-structures and their properties. Here, the self-assembly mechanism of a peptide-perylenediimide (P-1) conjugate in mixed solvent systems of THF/water is studied and the semiconducting properties are correlated with the morphology. In THF, right handed helical fibers are formed while in 10% THF-water, the morphology changes to nano-rings along with a switch in the helicity to left-handed orientation. Experimental results combined with DFT calculations reveal the critical role of thermodynamic and kinetic factors to control these differential self-assembly processes. In THF, P-1 forms right handed helical fibers in a kinetically controlled fashion. In case of 10% THF-water, the initial nucleation of the aggregate is controlled kinetically. Due to differential solubility of the molecule in these two solvents, elongation of the nuclei into fibers is restricted after a critical length leading to the formation of nano-rings which is governed by the thermodynamics. The helical fibers show superior semi-conducting property to the nano-rings as confirmed by conducting-AFM and conventional I-V characteristics.