Spectrophotometry and pharmaceutical PAT/RTRT: Practical challenges and regulatory landscape from development to product lifecycle.

European Pharmacopoeia includes dedicated chapters for Raman, NIR and Chemometrics, as well as there is a lot of Academia research on the matter. Despite that, the word innovation is often associated to such tools and there is a still slow implementation at industry. The paper is the outcome of the Associazione Farmaceutici dell'Industria (AFI) Study Group on Process Innovation and Product Lifecycle; the aim is to describe some case studies referring to practical approaches in pharmaceutical industry, in order to depict challenges and opportunities for the implementation of spectroscopic techniques. Case studies include: feasibility and pre-screening evaluations, chemometric model development approaches, way for the method maintenance during commercial manufacturing, challenges for implementation on existing equipment and on sterile processes. Case studies refer to oral solid products, liquid products and sterile Active Pharmaceutical Ingradient (API) manufacturing. There are already successful and robust spectroscopic applications in pharmaceutical industry and the technology is mature: this is the outcome of a strong applied research performed at pharmaceutical production departments. It is necessary to acknowledge efforts done by industry as Research for strengthening the cooperation with Academia, so that advantage of process innovation might reach the patients in a fastest way.

Atomic force microscopy analysis of cell volume regulation.

Cells swell in response a hypoosmotic challenge. By converting osmotic pressure to hydrostatic pressure at the cell membrane via van't Hoff's law, and converting that to tension via Laplace's law one predicts that the cell membrane should stretch and become stiff. We tested this prediction using the atomic force microscopy. During osmotic swelling cells did not become stiff and generally became softer. This result contradicts the assumption of the cell membrane as the constraining element in osmotic stress but is consistent with the cytoskeleton acting as a cross-linked gel. Models of the cells' response to osmotic stress must include energy terms for three-dimensional stresses.

Drift-free atomic force microscopy measurements of cell height and mechanical properties.

Atomic force microscopy (AFM) is used to study the morphological and mechanical properties of living cells. However, experiments performed over minutes to hours are subject to significant instrumental drift. The main sources of drift are the cantilever's geometrical asymmetry and bimorphic construction. We developed a simple software Stick-and-Move (SaM) routine for AFM that eliminates drift by continuously referencing the sample position to the substrate while acquiring force-distance curves. Control experiments show no drift over 15 min at an acquisition rate of 0.1 Hz. As a proof of concept, we applied the SaM to study the response of rat astrocytes to osmotic stress, observing dimensional and constitutive changes during volume regulation.

Hyaluronan conformations on surfaces: effect of surface charge and hydrophobicity.

Extended, relaxed, condensed, and interacting forms of the polysaccharide hyaluronan have been observed by atomic force microscopy (AFM). The types of images obtained depend on the properties of the surfaces used. We have investigated several different surface conditions for HA imaging, including unmodified mica, mica chemically modified with two different kinds of amino-terminated silanes (3-aminopropyltriethoxysilane and N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride), and highly oriented pyrolytic graphite. We found the degree of HA molecular extension or condensation to be variable, and the number of bound chains per unit area was low, for all of the mica-based surfaces. HA was more easily imaged on graphite, a hydrophobic surface. Chains were frequently observed in high degrees of extension, maintained by favorable interaction with the surface after molecular combing. This observation suggests that the HA macromolecule interacts with graphite through hydrophobic patches along its surface. AFM studies of HA behavior on differing surfaces under well-controlled environmental conditions provides useful insight into the variety of conformations and interactions likely to be found under differing physiological conditions.

Extended, relaxed, and condensed conformations of hyaluronan observed by atomic force microscopy.

The conformation of the polysaccharide hyaluronan (HA) has been investigated by tapping mode atomic force microscopy in air. HA deposited on a prehydrated mica surface favored an extended conformation, attributed to molecular combing and inhibition of subsequent chain recoil by adhesion to the structured water layer covering the surface. HA deposited on freshly cleaved mica served as a defect in a partially structured water layer, and favored relaxed, weakly helical, coiled conformations. Intramolecularly condensed forms of HA were also observed, ranging from pearl necklace forms to thick rods. The condensation is attributed to weak adhesion to the mica surface, counterion-mediated attractive electrostatic interactions between polyelectrolytes, and hydration effects. Intermolecular association of both extended and condensed forms of HA was observed to result in the formation of networks and twisted fibers, in which the chain direction is not necessarily parallel to the fiber direction. Whereas the relaxed coil and partially condensed conformations of HA are relevant to the native structure of liquid connective tissues, fully condensed rods may be more relevant for HA tethered to a cell surface or intracellular HA, and fibrous forms may be relevant for HA subjected to shear flow in tight intercellular spaces or in protein-HA complexes.

Imaging structured water and bound polysaccharide on mica surface at ambient temperature.

The presence of a water layer on the surface of muscovite mica under ambient conditions is well established. The water molecules are well ordered and seem to be oriented, leading to an icelike monolayer (probably ferroelectric) in epitaxial relation with the mica surface. We have imaged and characterized the height and contact angle of ordered water layer(s) formed by wetting and de-wetting processes on mica surfaces at different states of hydration by tapping mode atomic force microscopy. Implications that the presence of such an ordered water layer may have for imaging of biological samples are also discussed, with consideration of data for the polysaccharide hyaluronan.

Activity of Candida rugosa lipase immobilized on gamma-Fe2O3 magnetic nanoparticles.

We report the stability and enzymatic activity of Candida rugosa Lipase (E.C.3.1.1.3) immobilized on gamma-Fe2O3 magnetic nanoparticles. The immobilization strategies were either reacting the enzyme amine group with a nanoparticle surface acetyl, or amine groups. In the former, the enzyme was attached through a C=N bond, while in the latter it was connected using glutaraldehyde. AFM images show an average particle size of 20 +/- 10 nm after deconvolution. The enzymatic activity of the immobilized lipase was determined by following the ester cleavage of p-nitrophenol butyrate. The covalently immobilized enzyme was stabile and reactive over 30 days.

Poly(vinyl alcohol) as versatile biomaterial for potential biomedical applications.

In this paper, we present some new case examples where the chemical versatility of poly (vinyl alcohol) (PVA) can be used for potential biomedical applications. PVA, the polymeric material used for designing new nanostructured devices, is water soluble, biocompatible and has excellent physical properties. We point out the possibility of obtaining wall-to-wall chemical hydrogels as well as microgels without diminishing the biocompatibility available in the starting PVA material. Injectability is another important factor to take into account in controlled drug delivery for gene therapy. In this respect, in this paper, established and more innovative methods are prospected in order to obtain particles with dimensions suitable for these applications.