Folding Behaviors of Protein (Lysozyme) Confined in Polyelectrolyte Complex Micelle

The folding/unfolding behavior of proteins (enzymes) in confined space is important for their properties and functions, but such a behavior remains largely unexplored. In this article, we reported our finding that lysozyme and a double hydrophilic block copolymer, methoxy­poly­(ethylene glycol)_5K-<i>block</i>-poly­(l-aspartic acid sodium salt)₁₀ (mPEG_5K-<i>b</i>-PLD₁₀), can form a polyelectrolyte complex micelle with a particle size of ∼30 nm, as verified by dynamic light scattering and transmission electron microscopy. The unfolding and refolding behaviors of lysozyme molecules in the presence of the copolymer were studied by microcalorimetry and circular dichroism spectroscopy. Upon complex formation with mPEG_5K-<i>b</i>-PLD₁₀, lysozyme changed from its initial native state to a new partially unfolded state. Compared with its native state, this copolymer-complexed new folding state of lysozyme has different secondary and tertiary structures, a decreased thermostability, and significantly altered unfolding/refolding behaviors. It was found that the native lysozyme exhibited reversible unfolding and refolding upon heating and subsequent cooling, while lysozyme in the new folding state (complexed with the oppositely charged PLD segments of the polymer) could unfold upon heating but could not refold upon subsequent cooling. By employing the heating–cooling–reheating procedure, the prevention of complex formation between lysozyme and polymer due to the salt screening effect was observed, and the resulting uncomplexed lysozyme regained its proper unfolding and refolding abilities upon heating and subsequent cooling. Besides, we also pointed out the important role the length of the PLD segment played during the formation of micelles and the monodispersity of the formed micelles. Furthermore, the lysozyme–mPEG_5K-<i>b</i>-PLD₁₀ mixtures prepared in this work were all transparent, without the formation of large aggregates or precipitates in solution as frequently observed in other protein–polyelectrolyte systems. Hence, the present protein–PEGylated poly­(amino acid) mixture provides an ideal water-soluble model system to study the important role of electrostatic interaction in the complexation between proteins and polymers, leading to important new knowledge on the protein–polymer interactions. Moreover, the polyelectrolyte complex micelle formed between protein and PEGylated polymer may provide a good drug delivery vehicle for therapeutic proteins

Complexation of Lysozyme with Sodium Poly(styrenesulfonate) via the Two-State and Non-Two-State Unfoldings of Lysozyme

To provide an in-depth understanding of the complexation mechanism of protein and polyelectrolyte, a heating–cooling–reheating protocol was employed to study the unfolding and refolding behaviors of a model protein, lysozyme, in the presence of a negatively charged polyelectrolyte, sodium poly­(styrenesulfonate) (PSS). It was found that, with elevated PSS concentration, a new state (state I) was first formed via a “two-state” conversion process and this state could further convert to a completely unfolded state (state II) via a “non-two-state” conversion. This non-two-state conversion process occurs without the coexistence of states I and II but involves the formation of various intermediate unfolded protein structures. Different from the pure lysozyme that exhibited refolding upon cooling from its heat-denatured state, lysozyme in state I could undergo unfolding upon heating but no refolding upon cooling, while lysozyme in state II did not undergo unfolding or refolding upon thermal treatments. In addition, the effects of ionic strength and molecular weight of polyelectrolyte on the unfolding and refolding behaviors of lysozyme were also investigated. The present work provides a better understanding of the principles governing protein–polyelectrolyte interactions and may have implications for the fabrication of biocolloids and biofilms

In Situ Visualization of Lipid Raft Domains by Fluorescent Glycol Chitosan Derivatives

Lipid rafts are highly ordered small microdomains mainly composed of glycosphingolipids, cholesterol, and protein receptors. Optically distinguishing lipid raft domains in cell membranes would greatly facilitate the investigations on the structure and dynamics of raft-related cellular behaviors, such as signal transduction, membrane transport (endocytosis), adhesion, and motility. However, current strategies about the visualization of lipid raft domains usually suffer from the low biocompatibility of the probes, invasive detection, or ex situ observation. At the same time, naturally derived biomacromolecules have been extensively used in biomedical field and their interaction with cells remains a long-standing topic since it is closely related to various fundamental studies and potential applications. Herein, noninvasive visualization of lipid raft domains in model lipid bilayers (supported lipid bilayers and giant unilamellar vesicles) and live cells was successfully realized in situ using fluorescent biomacromolecules: the fluorescein isothiocyanate (FITC)-labeled glycol chitosan molecules. We found that the lipid raft domains in model or real membranes could be specifically stained by the FITC-labeled glycol chitosan molecules, which could be attributed to the electrostatic attractive interaction and/or hydrophobic interaction between the probes and the lipid raft domains. Since the FITC-labeled glycol chitosan molecules do not need to completely insert into the lipid bilayer and will not disturb the organization of lipids, they can more accurately visualize the raft domains as compared with other fluorescent dyes that need to be premixed with the various lipid molecules prior to the fabrication of model membranes. Furthermore, the FITC-labeled glycol chitosan molecules were found to be able to resist cellular internalization and could successfully visualize rafts in live cells. The present work provides a new way to achieve the imaging of lipid rafts and also sheds new light on the interaction between biomacromolecules and lipid membranes

Enhanced Radiosensitization of Gold Nanospikes via Hyperthermia in Combined Cancer Radiation and Photothermal Therapy

Metallic nanostructures as excellent candidates for nanosensitizers have shown enormous potentials in cancer radiotherapy and photothermal therapy. Clinically, a relatively low and safe radiation dose is highly desired to avoid damage to normal tissues. Therefore, the synergistic effect of the low-dosed X-ray radiation and other therapeutic approaches (or so-called “combined therapeutic strategy”) is needed. Herein, we have synthesized hollow and spike-like gold nanostructures by a facile galvanic replacement reaction. Such gold nanospikes (GNSs) with low cytotoxicity exhibited high photothermal conversion efficiency (η = 50.3%) and had excellent photostability under cyclic near-infrared (NIR) laser irradiations. We have demonstrated that these GNSs can be successfully used for in vitro and in vivo X-ray radiation therapy and NIR photothermal therapy. For the in vitro study, colony formation assay clearly demonstrated that GNS-mediated photothermal therapy and X-ray radiotherapy reduced the cell survival fraction to 89% and 51%, respectively. In contrast, the cell survival fraction of the combined radio- and photothermal treatment decreased to 33%. The synergistic cancer treatment performance was attributable to the effect of hyperthermia, which efficiently enhanced the radiosensitizing effect of hypoxic cancer cells that were resistant to ionizing radiation. The sensitization enhancement ratio (SER) of GNSs alone was calculated to be about 1.38, which increased to 1.63 when the GNS treatment was combined with the NIR irradiation, confirming that GNSs are effective radiation sensitizers to enhance X-ray radiation effect through hyperpyrexia. In vivo tumor growth study indicated that the tumor growth inhibition (TGI) in the synergistically treated group reached 92.2%, which was much higher than that of the group treated with the GNS-enhanced X-ray radiation (TGI = 29.8%) or the group treated with the GNS-mediated photothermal therapy (TGI = 70.5%). This research provides a new method to employ GNSs as multifunctional nanosensitizers for synergistic NIR photothermal and X-ray radiation therapy in vitro and in vivo

Glutathione-Depleting Gold Nanoclusters for Enhanced Cancer Radiotherapy through Synergistic External and Internal Regulations

The therapeutic performance of cancer radiotherapy is often limited by the overexpression of glutathione (GSH) in tumors and low radiation sensitivity of cancerous cells. To address these issues, the facilely prepared histidine-capped gold nanoclusters (Au NCs@His) were adopted as a radiosensitizer with a high sensitization enhancement ratio of ∼1.54. On one hand, Au NCs@His can inherit the local radiation enhancement property of gold-based materials (external regulation); on the other hand, Au NCs@His can decrease the intracellular GSH level, thus preventing the generated reactive oxygen species (ROS) from being consumed by GSH, and arrest the cells at the radiosensitive G2/M phase (internal regulation)