Lipid molecules such as glycolipids that are modified with hydrophilic biopolymers participate in the biochemical reactions occurring on cell membranes. Their functions and efficiency are determined by the formation of microdomains and their physical properties. We investigated the morphology and properties of domains induced by the hydrophilic-polymer-modified lipid applying a polyethylene glycol (PEG)-modified lipid as a model modified lipid. We formed supported lipid bilayers (SLBs) using a 0-10 mol % range of PEG-modified lipid concentration ( C). We studied their morphology and fluidity by fluorescence microscopy, the fluorescence recovery after photobleaching method, and atomic force microscopy (AFM). Fluorescence images showed that domains rich in the PEG-modified lipid appeared and SLB fluidity decreased when C ≥ 5%. AFM topographies showed that clusters of the PEG-modified lipid appeared prior to domain formation and the PEG-lipid-rich domains were observed as depressions. Frequency-modulation AFM revealed a force-dependent appearance of the PEG-lipid-rich domain.

Messenger RNA (mRNA) delivery strategies are required to protect biologically fragile mRNA from ribonuclease (RNase) attacks to achieve efficient therapeutic protein expression. To tackle this issue, most mRNA delivery systems have used cationic components, which form electrostatically driven complexes with mRNA and shield encapsulated mRNA strands. However, cationic materials interact with anionic biomacromolecules in physiological environments, which leads to unspecific reactions and toxicities. To circumvent this issue of cation-based approaches, herein, we propose a cation-free delivery strategy by hybridization of PEGylated RNA oligonucleotides with mRNA. The PEG strands on the mRNA sterically and electrostatically shielded the mRNA, improving mRNA nuclease stability 15-fold after serum incubation compared with unhybridized mRNA. Eventually, the PEGylated mRNA induced nearly 20-fold higher efficiency of reporter protein expression than unhybridized mRNA in cultured cells. This study provides a platform to establish a safe and efficient cation-free mRNA delivery system.

Eradication of cancer stem cells (CSCs) is an ultimate goal in cancer chemotherapy. Although a ligand-assisted targeting approach seems rational, the existence of subpopulations of CSCs and their discrimination from those present on healthy sites makes it a severe challenge. Some boronic acid (BA) derivatives are known for the ability to bind with glycan-terminal sialic acid (SA), in a manner dependent on the acidification found in hypoxic tumoral microenvironment. Taking advantage of this feature, here we show that the BA−ligand fluorescence conjugate can effectively target multiple CSC subpopulations in parallel, which otherwise must be independently aimed when using antibody−-ligands.

Macrocyclic drugs are promising agents for treating a variety of diseases. However, these compounds usually present delivery limitations, such as low tissue selectivity and poor cellular uptake, which may impair efficacy and clinical translation. Here, we propose a molecular machine approach for delivering macrocyclic drugs based on their assembly into bioactive rotaxanes. To prove this concept, we use the extremely toxic macrocycle valinomycin (Val) as the host molecule, and identify dihydralazine (Dihyd) as a guest molecule after screening several guest compounds. The Val-Dihyd complex is mechanically interlocked by capping one hydrazide group in Dihyd with fluorescein isothiocyanate (FITC) and the other with a Y-shape branched poly(ethylene glycol) (PEG) via a pH-sensitive hydrazone bond. Thus, the Val-loaded rotaxanes (Vrot) are stable at physiological pH, but release Val at mild acidic conditions mimicking intratumoral and endosomal environments. In vitro studies revealed Vrot is less cytotoxic than free Val in pancreatic cancer cells, while modifying Vrot with cyclic arginine-glycine-aspartic acid (cRGD) peptides promotes the cytotoxicity by enhancing cellular uptake. These results indicate the potential of rotaxanes of macrocyclic drugs for generating cancer targeted prodrugs.