Hyaluronan (HA) is a naturally occurring linear, negatively charged polysaccharide that plays a vital role in the organization and function of pericellular coats and extracellular matrices in vertebrates, and that is becoming increasingly popular in biomedical applications. To gain insight into the physical phenomena that govern the mechanical behavior of HA assemblies, we have studied the response of films of end-grafted HA to compression over a large range of ionic strength. Compression forces were measured as a function of the absolute distance between a colloidal probe and the planar surface on which the HA film was constructed, using a combined atomic force microscopy and reflection interference contrast microscopy setup. The HA films were well-defined in the sense that they are made of chains with a narrow size distribution that are grafted at controlled density to a solid support. Detailed comparison of the experimental data with analytical expressions derived from polymer and polyelectrolyte brush theory reveals that films of end-grafted HA behave as strongly charged polyelectrolyte brushes. To quantitatively reproduce the experimental data, intrinsic excluded volume interactions and chain stiffness of the polymer backbone must be taken into account. At low ionic strength, chains become almost fully stretched. In our experimental system, several micrometer thick films are formed that reach a hydration of up to 99.98%, and the brush thickness decreases by more than 5-fold with increasing ionic strength. More generally, the study provides quantitative theoretical predictions for the film thickness and compressive response as a function of HA length, grafting density and ionic strength. ■ INTRODUCTIONThe polysaccharide hyaluronan (HA) is ubiquitous in the pericellular and extracellular space of vertebrates. It is also becoming increasingly popular in biomedical applications as surface coating 1 and in tissue engineering. 2,3 HA is a linear polymer of disaccharides, made of glucuronic acid and Nacetylglucosamine, that are linked via alternating β-1,4 and β-1,3 glycosidic bonds. Each disaccharide has a length of 1 nm 4 and carries one chargeable group in the form of a carboxylic acid. For polymeric HA, the pK a is approximately 3, 5 and HA is, hence, negatively charged above pH ≈ 3. In vivo, HA is expressed by HA synthases at the cell membrane and extruded into the extracellular space. These HA molecules have a molecular mass of typically a few million Daltons 6 or a contour length of several micrometers. Once produced, they can stay grafted to the synthases and/or bind to other HA receptors at the cell surface, such as CD44 7 and form so-called pericellular coats (PCCs), or they can be released into solution to serve other functions in the extracellular space. Numerous biological functions of PCCs 8−11 and extracellular assemblies of HA in general (e.g., in cartilage) have been related to their mechanical properties. To better understand how the mechanical properties of PCCs are connected to their ...

We describe a method that combines colloidal probe atomic force microscopy (AFM) and reflection interference contrast microscopy (RICM) to characterize the mechanical properties of thin and solvated polymer films. When analyzing polymer films, a fundamental problem in colloidal probe AFM experiments is to determine the distance at closest approach between the probe and the substrate on which the film is deposited. By combining AFM and RICM in situ, forces and absolute distances can be measured simultaneously. Using the combined setup, we quantify the compressive mechanics of films of the polysaccharide hyaluronan that is end-grafted to a supported lipid bilayer. The experimental data, and comparison with polymer theory, show that hyaluronan films are well-described as elastic, very soft and highly solvated polymer brushes. The data on these well-defined films should be a useful reference for the investigation of the more complex hyaluronan-rich coats that surround many living cells.

Movements in animals arise through concerted action of neurons and skeletal muscle. General anaesthetics prevent movement and cause loss of consciousness by blocking neural function. Anaesthetics of the amino amide-class are thought to act by blockade of voltage-gated sodium channels. In fish, the commonly used anaesthetic tricaine methanesulphonate, also known as 3-aminobenzoic acid ethyl ester, metacaine or MS-222, causes loss of consciousness. However, its role in blocking action potentials in distinct excitable cells is unclear, raising the possibility that tricaine could act as a neuromuscular blocking agent directly causing paralysis. Here we use evoked electrical stimulation to show that tricaine efficiently blocks neural action potentials, but does not prevent directly evoked muscle contraction. Nifedipine-sensitive L-type Cav channels affecting movement are also primarily neural, suggesting that muscle Nav channels are relatively insensitive to tricaine. These findings show that tricaine used at standard concentrations in zebrafish larvae does not paralyse muscle, thereby diminishing concern that a direct action on muscle could mask a lack of general anaesthesia.