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.

Trade credit linkages along the supply chain: evidence for the Italian textile sector

We examine the trade credit linkages among firms within a supply chain to reckon the effect of such linkages on the propagation of liquidity shocks from downstream to upstream firms. We choose a sample appropriate for this task, consisting of a large data set of Italian firms from the textile industry, a well known example of a comprehensive manufacturing cluster featuring a large number of small and specialized firms at each level of the supply chain. The results of the analysis indicate that the level of trade credit that firms provide to their suppliers is positively related to the level of trade credit granted to their clients: when the level of trade credit granted to clients divided by sales goes up by 1, the level of trade credit provided to suppliers divided by cost-of goods-sold goes up by an amount that varies between 0,22 and 0,52. Since all firms along the chain are linked by trade credit relationships, an increase in the level of trade credit granted by wholesalers generates a liquidity cascade throughout the chain. We designate the overall increase in the level of trade credit among all firms in the chain as a result of a unitary impulse in the level of trade credit granted by wholesalers as the multiplier effect of trade credit for the industry chain. We estimate such multiplier to vary between 1.28 and 2.04. We also investigate the effect of final demand on the level of trade credit sourced by firms at various levels of the chain and, in particular, whether such effect is amplified for firms further up in the chain as a result of liquidity propagation via trade credit linkages. We uncover evidence of such amplification when the links of liquidity transmission along the chain are individually modeled and estimated. An unitary increase in wholesalers’ sales is found to produce an effect on trade payables among firms at the top of the chain (i.e., Preparers and Spinners) that is more than twice as big as the corresponding effect among firms at the bottom of the chain (i.e., Wholesalers).

Learning and practising supply chain management strategies from a business simulation game: a comprehensive supply chain simulation

An Internet based supply chain simulation game (ISCS) is introduced and demonstrated in this paper. Different from other games and extended from the Beer Game, a comprehensive set of supply chain (SC) management strategies can be tested in the game, and these strategies can be evaluated and appraised based on the built-in Management Information System (MIS). The key functionalities of ISCS are designed to increase players SC awareness, facilitate understanding on various SC strategies and challenges, foster collaboration between partners, and improve problem solving skills. It is concluded that an ISCS can be used as an efficient and effective teaching tool as well as a research tool in operations research and management science. Problems and obstacles have been observed while engaging in the SC business scenario game. The actions proposed and implemented to solve these problems have resulted in improved SC performance.

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.

Development and mechanical characterization of solvent-cast polymeric films as potential drug delivery systems to mucosal surfaces

Solvent-cast films from three polymers, carboxymethylcellulose (CMC), sodium alginate (SA), and xanthan gum, were prepared by drying the polymeric gels in air. Three methods, (a) passive hydration, (b) vortex hydration with heating, and (c) cold hydration, were investigated to determine the most effective means of preparing gels for each of the three polymers. Different drying conditions [relative humidity - RH (6-52%) and temperature (3-45 degrees C)] were investigated to determine the effect of drying rate on the films prepared by drying the polymeric gels. The tensile properties of the CMC films were determined by stretching dumbbell-shaped films to breaking point, using a Texture Analyser. Glycerol was used as a plasticizer, and its effects on the drying rate, physical appearance, and tensile properties of the resulting films were investigated. Vortex hydration with heating was the method of choice for preparing gels of SA and CMC, and cold hydration for xanthan gels. Drying rates increased with low glycerol content, high temperature, and low relative humidity. The residual water content of the films increased with increasing glycerol content and high relative humidity and decreased at higher temperatures. Generally, temperature affected the drying rate to a greater extent than relative humidity. Glycerol significantly affected the toughness (increased) and rigidity (decreased) of CMC films. CMC films prepared at 45 degrees C and 6% RH produced suitable films at the fastest rate while films containing equal quantities of glycerol and CMC possessed an ideal balance between flexibility and rigidity.