The use of fluorescence microscopy to define polymer localisation to the late endocytic compartments in cells that are targets for drug delivery

Macromolecular therapeutics and nano-sized drug delivery systems often require localisation to specific intracellular compartments. In particular, efficient endosomal escape, retrograde trafficking, or late endocytic/lysosomal activation are often prerequisites for pharmacological activity. The aim of this study was to define a fluorescence microscopy technique able to confirm the localisation of water-soluble polymeric carriers to late endocytic intracellular compartments. Three polymeric carriers of different molecular weight and character were studied: dextrin (Mw~50,000 g/mol), a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (Mw approximately 35,000 g/mol) and polyethylene glycol (PEG) (Mw 5000 g/mol). They were labelled with Oregon Green (OG) (0.3-3 wt.%; <3% free OG in respect of total). A panel of relevant target cells were used: THP-1, ARPE-19, and MCF-7 cells, and primary bovine chondrocytes (currently being used to evaluate novel polymer therapeutics) as well as NRK and Vero cells as reference controls. Specific intracellular compartments were marked using either endocytosed physiological standards, Marine Blue (MB) or Texas-red (TxR)-Wheat germ agglutinin (WGA), TxR-Bovine Serum Albumin (BSA), TxR-dextran, ricin holotoxin, C6-7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labelled ceramide and TxR-shiga toxin B chain, or post-fixation immuno-staining for early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins (LAMP-1, Lgp-120 or CD63) or the Golgi marker GM130. Co-localisation with polymer-OG conjugates confirmed transfer to discreet, late endocytic (including lysosomal) compartments in all cells types. The technique described here is a particularly powerful tool as it circumvents fixation artefacts ensuring the retention of water-soluble polymers within the vesicles they occupy.

Ion release by endodontic grade glass-ionomer cement

Cylindrical specimens (6 mm high x 4 mm diameter) of the endodontic grade glass-ionomer (Ketac Endo) were exposed to various media for 1 week, after which changes in their mass, pH of storage medium, and ion release were determined. In water, this cement was shown to release reasonable amounts of sodium, aluminium and silicon, together with smaller amounts of calcium and phosphorus, as well as taking up 2.41% by mass of water. A comparison with the restorative grade materials (Ketac Molar, ex 3M ESPE and Fuji IX, ex GC) showed both ion release and water uptake to be greater. All three cements shifted pH from 7 to around 6 with no significant differences between them. Other storage media were found to alter the pattern of ion release. Lactic acid caused an increase, whereas both saturated calcium hydroxide and 0.6% sodium hypochlorite, caused decreases. This suppression of ion-release may be significant clinically. Aluminium is the most potentially hazardous of the ions involved but amounts released were low compared with levels previously reported to show biological damage.

Investigation of wall-slip effect on paste release characteristic in flip-chip stencil printing process

As the trend toward further miniaturisation of pocket and handheld consumer electronic products continues apace, the requirements for even smaller solder joints will continue. With further reductions in the size of solder joints, the reliability of solder joints will become more and more critical to the long-term performance of electronic products. Solder joints play an important role in electronics packaging, serving both as electrical interconnections between the components and the board, and as mechanical support for components. With world-wide legislation for the removal/reduction of lead and other hazardous materials from electrical and electronic products, the electronics manufacturing industry has been faced with an urgent search for new lead-free solder alloy systems and other solder alternatives. In order to achieve high volume, low cost production, the stencil printing process and subsequent wafer bumping of solder paste has become indispensable. There is wide agreement in industry that the paste printing process accounts for the majority of assembly defects, and most defects originate from poor understanding of the effect of printing process parameters on printing performance. The printing of ICAs and lead-free solder pastes through the very small stencil apertures required for flip chip applications was expected to result in increased stencil clogging and incomplete transfer of paste to the printed circuit pads. Paste release from the stencil apertures is dependent on the interaction between the solder paste, surface pad and aperture wall; including its shape. At these very narrow aperture sizes the paste rheology becomes crucial for consistent paste withdrawal because for smaller paste volumes surface tension effects become dominant over viscous flow. Successful aperture filling and release will greatly depend on the rheology of the paste material. Wall-slip plays an important role in characterising the flow behaviour of solder paste materials. The wall- slip arises due to the various attractive and repulsive forces acting between the solder particles and the walls of the measuring geometry. These interactions could lead to the presence of a thin solvent layer adjacent to the wall, which gives rise to slippage. The wall slip effect can play an important role in ensuring successful paste release after the printing process. The aim of this study was to investigate the influence of the paste microstructure on slip formation for the paste materials (lead-free solder paste and isotropic conductive adhesives). The effect of surface roughness on the paste viscosity was investigated. It was also found that altering the surface roughness of the parallel plate measuring geometry did not significantly eliminate wall slip as was expected. But results indicate that the use of a relatively rough surface helps to increase paste adhesion to the plates, inducing structural breakdown of the paste. Most importantly, the study also demonstrated on how the wall slip formation in the paste material could be utilised for understanding of the paste microstructure and its flow behaviour

In vitro drug release studies of polymeric freeze-dried wafers and solvent-cast films using paracetamol as a model soluble drug

Drug dissolution and release characteristics from freeze-dried wafers and solvent-cast films prepared from sodium carboxymethylcellulose (CMC) have been investigated to determine the mechanisms of drug release from the two systems. The formulations were prepared by freeze-drying (wafers) or drying in air (films), the hydrated gel of the polymer containing paracetamol as a model soluble drug. Scanning electron microscopy (SEM) was used to examine differences between the physical structure of the wafers and films. Dissolution studies were performed using an exchange cell and drug release was measured by UV spectroscopy at 242 nm. The effects of drug loading, polymer content and amount of glycerol (films) on the release characteristics of paracetamol were investigated. The release profiles of paracetamol from the wafers and films were also compared. A digital camera was used to observe the times to complete hydration and dissolution of the wafers containing different amounts of CMC and how that impacts on drug release rates. Both formulations showed sustained type drug release that was modelled by the Korsmeyer–Peppas equation. Changes in the concentration of drug and glycerol (films) did not significantly alter the rate of drug release while increasing polymer content significantly decreased the rate of drug release from both formulations. The results show that the rate of paracetamol release was faster from the wafers than the corresponding films due to differences in their physical structures. The wafers which formed a porous network, hydrated faster than the more dense and continuous, (non-porous) sheet-like structure of the films.