Metal Ion-Induced Unusual Stability of the Metastable Vesicle-like Intermediates Evolving during the Self-Assembly of Phenylalanine: Prominent Role of Surface Charge Inversion.

The underlying mechanism and intermediate formation in the self-assembly of aromatic amino acids, peptides, and proteins remain elusive despite numerous reports. We, for the first time, report that one can stabilize the intermediates by tuning the metal ion-amino acid interaction. Microscopic and spectroscopic investigations of the self-assembly of carboxybenzyl (Z)-protected phenylalanine (ZF) reveal that the bivalent metal ions eventually lead to the formation of fibrillar networks similar to blank ZF whereas the trivalent ions develop vesicle-like intermediates that do not undergo fibrillation for a prolonged time. The time-lapse measurement of surface charge reveals that the surface charge of blank ZF and in the presence of bivalent metal ions changes from a negative value to zero, implying unstable intermediates leading to the fibril network. Strikingly, a prominent charge inversion from an initial negative value to a positive value in the presence of trivalent metal ions imparts unusual stability to the metastable intermediates.

Effect of Lipid Corona on Phenylalanine-Functionalized Gold Nanoparticles to Develop Stable and Corona-Free Systems.

Conventional gold nanoparticles (Au NPs) have many limitations, such as aggregation and subsequent precipitation in the medium of high ionic strength and protein molecules. Furthermore, when exposed to biological fluids, nanoparticles form a protein corona, which controls different biological processes such as the circulation lifetime, drug release profile, biodistribution, and in vivo cellular distribution. These limitations reduce the functionality of Au NPs in targeted delivery, bioimaging, gene delivery, drug delivery, and other biomedical applications. To circumvent these problems, there are numerous attempts to design corona-free and stable nanoparticles. Here, we report for the first time that lipid corona (coating of lipid) formation on phenylalanine-functionalized Au NPs (AuPhe NPs) imparts excellent stability against the high ionic strength of bivalent metal ions, amino acids, and proteins of different charges as compared to bare nanoparticles. Moreover, this work is focused on the ability of lipid corona formation on AuPhe NPs to prevent protein adsorption in the presence of cell culture medium (CCM), oppositely charged protein (e.g., histone 3), and human serum albumin (HSA). The results demonstrate that the lipid corona successfully protects the AuPhe NPs from protein adsorption, leading to the development of corona-free character. This unique achievement has profound implications for enhancing the biomedical utility and safety of these nanoparticles.

Copper-Catalyzed Regio- and Stereoselective Hydroarylation of Ynamide.

Presented herein is a copper-catalyzed trans-hydroarylation of ynamides. The reaction showcases the assembly of boronic acids across the carbon-carbon triple bond of ynamides. The reaction proceeds under mild conditions offering a complementary approach for the versatile synthesis of multifunctional (E)-α,ß-disubstituted enamides. Moreover, the hydroarylation process is highly regio- and stereoselective. The transformation shows a broad scope (30 examples) and tolerates a wide range of labile functional groups. Control experiments provide substantive evidence supporting the mechanistic cycle and the observed selectivity.

Insight into the Lysozyme-Induced Aggregation of Aromatic Amino Acid-Functionalized Gold Nanoparticles: Impact of the Protein Conjugation and Lipid Corona on the Aggregation Phenomena.

The aggregation and subsequent precipitation of gold nanoparticles (Au NPs) in the presence of protein molecules restrict the usefulness of NPs in biomedical applications. Till now, the influence of different properties of Au NPs (size, surface charge, surface coatings) and proteins (surface charge, chemical modification, folded and unfolded states) and pH and ionic strength of the solution on the aggregation of both Au NPs and proteins has been thoroughly discussed in the literature. However, the underlying different mechanistic pathways of the protein concentration-dependent aggregation of both Au NPs and proteins are poorly understood. The impact of the lipid corona on the protein-induced Au NP aggregation has remained an unresolved issue. In this context, we investigate the interaction of the negatively charged aromatic amino acid (phenylalanine and tyrosine)-functionalized gold nanoparticles (Au-AA NPs) with the positively charged globular protein lysozyme at different protein concentrations and compare the results with those of conventional citrate-functionalized Au NPs (Au-Cit NPs). Next, we conjugate lipids and proteins to Au NPs to impede the aggregation of Au NPs induced by the lysozyme. Our results reveal that the aggregation mechanism of the Au-AA NPs is distinctly different at low and high protein concentrations with the uniqueness of the Au-AA NPs over the Au-Cit NPs. Furthermore, we find that human serum albumin (HSA) protein-conjugated Au-AA and Au-Cit NPs are more effective in preventing the lysozyme-induced Au NP aggregation than bovine serum albumin (BSA)-conjugated Au NPs. For the first time, we also report the significant role of "hard" and "soft" lipid coronas in the aggregation of amino acid (phenylalanine)-functionalized gold nanoparticles in the presence of lysozyme protein.

Metal-Ion-Induced Evolution of Phenylalanine Self-Assembly: Structural Polymorphism of Novel Metastable Intermediates.

The self-assembly of aromatic amino acids has been widely studied due to their ability to form well-defined amyloid-like fibrillar structures. Herein, for the first time, we report the existence of different metastable intermediate states of diverse morphologies, for example, droplets, spheres, vesicles, flowers, and toroids, that are sequentially formed in aqueous medium during the self-assembly process of phenylalanine in the presence of different divalent (Zn2+, Cd2+, and Hg2+) and trivalent (Al3+, Ga3+, and In3+) metal ions having low pKa values. Due to metal ion-amino acid coordination and strong hydrophobic interaction induced by these metal ions, spherical aggregates are obtained at the initial stage of the structural evolution and further transformed into other intermediate states. Our work may facilitate understanding of the role of metal ions in the amino acid self-assembly process and broaden future applications of the obtained nanostructures in drug delivery, tissue engineering, bioimaging, biocatalysis, and other fields.

Mechanistic Pathway of Lipid Phase-Dependent Lipid Corona Formation on Phenylalanine-Functionalized Gold Nanoparticles: A Combined Experimental and Molecular Dynamics Simulation Study.

In recent years, the underlying mechanism of formation of the lipid corona and its stability have begun to garner interest in the nanoscience community. However, until now, very little is known about the role of different properties of nanoparticles (NPs) (surface charge density, hydrophobicity, and size) in lipid corona formation. Apart from the physicochemical properties of NPs, the different properties of lipids remain elusive in lipid corona formation. In the present contribution, we have investigated the interaction of phenylalanine-functionalized gold NPs (Au-Phe NPs) with different zwitterionic lipid vesicles of different phase states (sol-gel and liquid crystalline at room temperature) as a function of lipid concentration. The main objective of the present work is to understand how the lipid phase affects lipid corona formation and lipid-induced aggregation in various media. Our results establish that the lipid phase state, area per lipid head group, and the buffer medium play important roles in lipid-induced aggregation. The lipid corona occurs for NPs at high lipid concentration, irrespective of the phase states and area per lipid head group of the lipid bilayer. Notably, the lipid corona also forms at a low concentration of lipid vesicles in the liquid crystalline phase (1,2-dioleoyl-sn-glycero-3-phosphocholine). The corona formation brings in remarkable stability to NPs against freeze-thaw cycles. Based on the stability, for the first time, we classify lipid corona as "hard lipid corona" and "soft lipid corona". This distinct classification will help to develop suitable nanomaterials for various biomedical applications.

Effect of Metal Ions on the Intrinsic Blue Fluorescence Property and Morphology of Aromatic Amino Acid Self-Assembly.

Metal ions are known to strongly bind with different proteins and peptides, resulting in alteration of their different physicochemical properties. In this work, we investigate the effect of metal ions of different nuclear charges and sizes on the intrinsic blue luminescence of the self-assembled structures formed by aromatic amino acids, namely, phenylalanine and tryptophan, using spectroscopic and imaging techniques. The study reveals that the intrinsic blue fluorescence of amino acid assemblies is influenced by metal ions and the pH of the medium. The metal ions with a higher charge to radius ratio promote clusterization which results in the enhancement of the intrinsic fluorescence, an effect known as "clusteroluminescence" of the amino acids aggregates. The imaging study reveals that metal ions with a higher charge to size ratio inhibit the large fibrillation of aromatic amino acids by promoting the formation of small nonfibrillar aggregates through increased hydrophobicity in the medium. The nanoaggregates are assumed to be responsible for the enhancement in the blue "clusteroluminescence".

Lipid phase dependent distinct emission behaviour of hydrophobic carbon dots: C-dot based membrane probes.

We observe a unique distinct emission behaviour of hydrophobic carbon dots (H-CDs) embedded within the ordered and the disordered phase of a lipid membrane. The H-CDs exhibit blue emission in the disordered phase, however, they exhibit an intense red emission in the ordered phase of the lipid bilayer. The H-CDs have the potential ability to probe membrane dynamics like previously reported organic dyes. To the best of our knowledge, this is the first report of a CD-based membrane probe.

Underlying Mechanisms for the Modulation of Self-Assembly and the Intrinsic Fluorescent Properties of Amino Acid-Functionalized Gold Nanoparticles.

The origin of the blue fluorescence of proteins and peptides in the visible region has been a subject of intense debate despite several efforts. Although aromatic amino acids, namely tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) are responsible for the intrinsic luminescence of proteins and peptides, the underlying mechanism and contributions of these amino acids to the unusual blue fluorescence are still not well resolved. In the present endeavor, we show that the clusterization of both aromatic and aliphatic amino acids on the surface of the gold nanoparticles (Au NPs) leads to clusteroluminescence, which could be linked to the unusual fluorescence properties of the proteins and peptides and have been ignored in the past. The amino acid monomers initially form small aggregates through clusterization, which provides the fundamental building blocks to establish the amyloid structure as well as the luminescence property. Because of the clusterization, these Au NPs/nano-aggregate systems are also found to exhibit remarkable stability against the freeze-thaw cycle and several other external stimuli, which can be useful for biological and biomedical applications.

Interaction of Aromatic Amino Acid-Functionalized Gold Nanoparticles with Lipid Bilayers: Insight into the Emergence of Novel Lipid Corona Formation.

The coating of proteins and lipids around the surface of the nanoparticles is known as "protein corona" and "lipid corona", respectively, which have promising biomedical applications. While protein corona formation is well-known, the lipid corona is relatively new and its stability is yet to be explored. In the present contribution, we report a novel lipid corona formation and its underlying mechanism using aromatic amino acid-functionalized gold nanoparticles (Au-AA NPs) as a template by means of spectroscopic (steady-state UV-visible and fluorescence) and imaging (CLSM, HR-TEM, and AFM) techniques. Our study demonstrates that in the presence of high lipid concentration Au-AA NPs intrinsically tow the lipid molecules from the lipid vesicles and decorate themselves by lipid leading to unique lipid corona formation. In contrast, at low lipid concentration Au-AA NPs undergo lipid-induced aggregation. The lipid-nanoparticle interaction is a time-dependent phenomenon and depends on the surface charge of both the lipid and the Au-AA NPs. The HR-TEM analysis indicates that the partial lipid coating is an intermediate step of lipid-induced aggregation and lipid corona formation of the Au-AA NPs. Significantly, we found that the colloidal property of these lipid-coated nanoparticles (lipid corona) is immune to resist extreme harsh conditions, that is, high acidic pH, several repetitive freeze-thaw cycles, and high salt concentration. The extra stability of Au-AA NPs upon the formation of lipid corona allows us to introduce new engineered nanoparticles for future prospective.

Interaction of aliphatic amino acids with zwitterionic and charged lipid membranes: hydration and dehydration phenomena.

In the present contribution, we investigate the interactions of lipid bilayer membranes of different charges and different phase states with aliphatic amino acids of varying charge (aspartic acid, glutamic acid, arginine and lysine) and hydrophobicity (serine, leucine and valine) by steady state and time-resolved spectroscopic techniques, dynamic light scattering (DLS) measurements and confocal imaging (CLSM). The study reveals that negatively charged amino acids such as aspartic acid and glutamic acid interact strongly with the lipid membranes particularly with negatively charged lipid membranes by stabilizing their gel phase. On the other hand, positively charged amino acids bring in hydration in the membranes. We explain this unique observation by the shift in pKa of amino acids in the vicinity of the lipid membranes and solvation and desolvation processes in the light of recent computer simulations. We also find that hydrogen bonding plays a significant role in governing the interaction of aliphatic amino acids with zwitterionic lipid membranes. The more polar serine bearing a hydroxyl group at the terminal carbon offers a stronger interaction with the lipid bilayer membranes as compared to its analogues leucine and valine, which are hydrophobic in nature.