Macromolecular crowding has a profound impact on the conformational dynamics and intermolecular interactions of biological macromolecules. In this context, the role of inert synthetic crowders in the protein−protein interactions of globular proteins is poorly understood. Here, using native human serum albumin (HSA) under physiological conditions, we show that macromolecular crowding induces liquid−liquid phase separation (LLPS) via liquid-like membrane-less droplet formation in a concentration-and time-dependent manner. Circular dichroism measurements reveal significant alteration in the secondary structure of HSA inside the droplet during aging. In contrast, at a high protein concentration, a liquid-to-solid-like phase transition has been observed upon maturation. Our findings reveal that the LLPS of HSA is mainly driven by enthalpically controlled intermolecular protein−protein interactions via hydrophobic contacts involving aromatic and/or nonaromatic residues. Moreover, modulation of LLPS of HSA has been demonstrated upon denaturation and ligand binding. This study highlights the importance of soft protein−protein interactions of globular proteins in a crowded cellular environment in driving the LLPS.

Photostable and inherently luminescent biocompatible nanomaterials have tremendous importance in biomedical research. Herein, we have designed a pH-responsive inherently luminescent carbon dot (CD)-based coacervate nanodroplet (ND) in the presence of cationic polymer poly­(diallyldimethyl­ammonium chloride) (PDADMAC). The physicochemical and photoluminescence (PL) properties of these NDs have been explored as a function of equilibration time (1 and 18 h), pH (2–12), and ionic strength (1–1000 mM NaCl) by using various spectroscopic and microscopic techniques. Equilibrating the binary mixture of CD and PDADMAC for 1 and 18 h results in the formation of smaller (73.0 ± 2.3 nm) and larger sized (∼400 nm) NDs (SNDs and LNDs), respectively. While these NDs are stable in a broad pH range (5–12) and low ionic strength (<200 mM) medium, they disassembled in lower acidic pH (<5.5) and a high ionic strength (>200 mM) medium. Using UV–vis and confocal laser scanning microscopy, we have demonstrated that these negatively charged (ζ-potential = −17.0 ± 0.5 mV) NDs can spontaneously sequester neutral, cationic, and anionic dyes inside their porous nanostructure without any aggregation or structural disruption. In addition, a high partition coefficient of 10.6 ± 1.1 has been estimated for the cationic anticancer drug doxorubicin toward these NDs. The cell viability assay of SNDs with kidney fibroblast cell lines (BHK-21) reveals excellent biocompatibility. Finally, the bare and ethidium bromide (EtBr)-loaded SNDs have been utilized toward time-dependent cellular uptake experiments. Our findings indicate that the internalized SNDs undergo disassembly at lower acidic pH of late endosomes/lysosomes. While released EtBr in the cytosol specifically bind with nucleic acid within the cell nucleus without losing their affinity, free CDs on the other hand stain the whole cell without any specificity. Taken together, our present findings highlight the potential of these biocompatible inherently luminescent pH-responsive NDs toward theranostic applications.

Cellular crowding plays a key role in regulating the enzymatic reactivity in physiological conditions, which is challenging to realize in the dilute phase. Enzymes drive a wide range of complex metabolic reactions with high efficiency and selectivity under extremely heterogeneous and crowded cellular environments. However, the molecular interpretation behind the enhanced enzymatic reactivity under a crowded milieu is poorly understood. Herein, using the horseradish peroxidase (HRP) and glucose oxidase (GOx) cascade pair, we demonstrate for the first time that macromolecular crowding induces liquid–liquid phase separation (LLPS) via the formation of liquid-like condensates/droplets and thereby increases the intrinsic catalytic efficiencies of HRP and GOx. Both these enzymes undergo crowding induced homotypic LLPS via enthalpically driven multivalent electrostatic as well as hydrophobic interactions. Using a set of kinetic and microscopic experiments, we show that precise synchronization of spontaneous LLPS and enzymatic transformations is key to realize the enhanced enzymatic activity under the crowded environments. Our findings reveal an unprecedented enhancement (91- to 205-fold) in the catalytic efficiency (k cat/K m) of HRP at pH 4.0 within the droplet phase relative to that in the bulk aqueous phase in the presence of different crowders. In addition, we have shown that other enzymes also undergo spontaneous LLPS under macromolecular crowding, signifying the generality of this phenomenon under the crowded environments. More importantly, coalescence driven highly regulated GOx/HRP cascade reactions within the fused droplets have been demonstrated with enhanced activity and specificity under the crowded environments. The present discovery highlights the active role of membraneless condensates in regulating the enzymatic efficacy for complex metabolic reactions under the crowded cellular environments and may find significant importance in the field of biocatalysis.

Nature utilizes cellular and subcellular compartmentalization to efficiently drive various complex enzymatic transformations via spatiotemporal control. In this context, designing of artificial nanoreactors for efficient catalytic transformations finds tremendous importance in recent times. One key challenge remains the design of multiple catalytic centers within the confined space of a nanoreactor without unwanted agglomeration and accessibility barrier for reactants. Herein, we report a unique blend of nanoscience and chemical catalysis using a metal-free hybrid synthetic protocell as a catalytic nanoreactor for redox and photocatalytic transformations, which are otherwise incompatible in bulk aqueous medium. Hybrid coacervate nanodroplets (NDs) fabricated from 2.5 nm-sized carbon dots (CDs) and poly­(diallyldimethyl)­ammonium chloride have been utilized toward reductive hydrogenation of nitroarenes in the presence of sodium borohydride (NaBH4). It has been found that the reduction mechanism follows the classical Langmuir–Hinshelwood (LH) model at the surface of embedded CDs inside the NDs via the generation of reactive surface hydroxyl groups. These NDs show excellent recyclability without any compromise on reaction kinetics and conversion yield. Importantly, spatiotemporal control over the hydrogenation reaction has been achieved using two mixed populations of coacervates. Moreover, efficient visible light-induced photoredox conversion of ferricyanide to ferrocyanide and artificial peroxidase-like activity have also been demonstrated inside these catalytic NDs. Our findings indicate that the individual polymer-bound CD inside the NDs acts as the catalytic center for both the redox and photocatalytic reactions. The present study highlights the unprecedented catalytic activity of the metal-free CD-based coacervate NDs and paves the way for next-generation catalytic nanoreactors for a wide range of chemical and enzymatic transformations.

Stimuli-responsive water-dispersible and structurally robust nanoassemblies find tremendous importance in the biomedical domain for delivery of therapeutically active hydrophobic drugs and bioimaging applications. Herein we have demonstrated the loading of a hydrophobic model anticancer drug, [(p-cymene)Ru(curcuminato)-Cl] (Ru-Cur), inside hydrophobic compartments of different nanoassemblies, namely, micelles, liposomes, and coacervate nanodroplets, and studied the in vitro pH-and temperature-dependent controlled-release profiles. In the present study, both carbon-dot and adenosine triphosphate (ATP)-based coacervate nanodroplets have been fabricated in the presence of poly(diallyldimethylammonium chloride) (PDADMAC). It has been observed that the coacervate nanodroplets provide an ideal microenvironment for efficient loading (loading content = 31.2%, and encapsulation efficiency = 99.6%) and sustained release of the hydrophobic drug. The tailorability in the structure and physicochemical properties of coacervate nanodroplets along with high drug loading and negligible drug leakage at physiological conditions makes them ideal nanocarriers over other conventional nanoassemblies. Experimental release profiles for Ru-Cur-loaded ATP nanodroplets at different pH values fit well with a semiempirical power law model. The fitted parameters reveal diffusion-and swelling-controlled-release mechanism in the pH range between 7.4 and 6 and diffusion-and erosion-controlled-release mechanism at pH 5. Moreover, it has been found that the temperature has a profound influence on the drug-release profiles. The present study provides fundamental insight into the pHresponsive disassembly mechanism and highlights the potential importance of these Ru-Cur-loaded coacervates toward various theranostic applications.