Could albumin affect the self-assembling properties of a block co-polymer system and drug release? An in-vitro study.

PURPOSE: This work investigated the influence of a model protein, bovine serum albumin (BSA), on the properties of a thermogelling formulation intended for administration inside body compartments where there is high albumin content, as in the case of inflamed joints; it also explored the relation between the variation of these properties and release performance of methotrexate (MTX), a drug used to treat forms of arthritis and rheumatic conditions. METHODS: The influence of BSA on the micellisation and gelation behaviour of Poloxamer 407, chosen as a model copolymer, was studied by differential scanning calorimetry (microDSC), dynamic light scattering (DLS), fluorescence spectroscopy and rheology studies. A release study of MTX loaded inside the hydrogel in presence and in absence of BSA was performed. RESULTS: DLS and microDSC data revealed that the micellisation process was not affected by the protein, as demonstrated by unaltered micellar size and thermodynamic parameters. While the presence of BSA in the copolymer system reduced gel consistency, the hydrogel release performance was only slightly affected. CONCLUSION: Our results suggested that the kinetics of MTX release mainly depended on the presence of the thermogelling copolymer, although other mechanisms related to BSA could be involved. Finally, the study assessed the feasibility of using a thermogelling hydrogel for in situ drug administration in areas with the presence of high protein concentrations.

Free energy changes and components implicit in the MWC allosteric model for the cooperative oxygen binding of hemoglobin.

Hill's plots of oxygen binding isotherms reveal the presence of a transition between two different oxygen affinities at the beginning and end of the isotherm. They correspond to the two conformations anticipated by the MWC model, namely, the T and R conformations at the beginning and end of oxygen binding, when the lower affinity of the T form develops into the higher affinity of the R form. The difference between the binding Gibbs free energy changes of the two affinities (Δ G(L)) is the free energy of binding cooperativity. Notably, Δ G(L) is positive in favor of the T form, which moves to a higher energy level upon oxygen release. Osmotic stress reveals a higher volume/surface ratio of deoxyhemoglobin, with a positive Δ G(W) also in favor of the T form. An increasing protein concentration shifts the isotherms to the right, indicating the formation of intermediate polymeric forms. The enthalpy of the intermediates shows a strong absorption of heat at the third oxygenation step because of polymer formation with quinary, and higher-order, structures. The disassembly of intermediate polymers releases energy with a negative Δ G that compensates and allows the positive values of Δ G(L). High-energy polymers are the barrier preventing the relaxation of the T and R conformations into one another. The MWC allosteric model is the best justification of oxygen binding cooperativity.

Effect of hydroxypropyl beta-cyclodextrin on the self-assembling and thermogelation properties of Poloxamer 407.

This paper deals with the rheological and thermal characterisation of the self-assembling behaviour of different Poloxamer 407 systems (15-30%, w/v), both alone or after the addition of various amounts of hydroxypropyl beta-cyclodextrin (5-20%, w/v). The knowledge of the interactions existing between the two kinds of molecules could allow the development of systems for parenteral administration containing also proteins or peptides drugs. A rheology (temperature sweep and frequency sweep test) study has been performed to characterise the thermogelation process while thermal analysis (nanoDSC) allowed the determination of both micellization and sol/gelation transition processes. These two techniques were also utilised to outline the variation in the critical micelle temperature (cmT) or in the sol/gel temperature (sgT,) of the systems containing also hydroxypropyl beta-cyclodextrin (HP beta-CD). Both the rheology and thermal analysis showed the presence of interesting interactions between the HP beta-CD and the Poloxamer 407, which cause a shift of both cmT and sgT. The presence of HP beta-CD modified also Poloxamer samples elastic characters and microrheological structure as demonstrated by the G'-G'' mechanical spectra. Rheological and thermal results outlined how these new systems could open the possibility to join the thermogelling behaviour offered by Poloxamer 407 with the well-known stabilization and protection ability of HP beta-CD, which could make possible the formulation of systems for the parenteral delivery of peptides and proteins.

Water-in-Oil Microemulsions for Protein Delivery: Loading Optimization and Stability.

BACKGROUND: Microemulsions are attractive delivery systems for therapeutic proteins and peptides due to their ability to enhance bioavailability. Although different proteins and peptides have been successfully delivered through such ternary systems, no information can be found about protein loading and the formulation stability when such microemulsions are prepared with pharmaceuticallyapproved oils and surfactants. The aim of this work was to optimise a ternary system consisting of water/ ethyl oleate/Span® 80-Tween® 80 and to determine its protein loading capacity and stability, using bovine serum albumin (BSA) as a model of biomolecule. METHODS: The optimization was carried out using a Central Composite Design and all the prepared formulations were characterised through dynamic light scattering, rheology, optical and polarized microscopy. Subsequently, the maximum loading capacity was determined and the stability of the final microemulsion with the highest content of protein was followed over six months. To investigate the structural features of the protein, BSA was recovered from the microemulsion and analysed through fluorescence spectroscopy. RESULTS: After incorporation of the protein in the microemulsion, a decrease of its aqueous solubility was observed. However, the formulation remained stable over six months and the native-like state of the recovered protein was demonstrated by fluorescence spectroscopy Conclusion: This study demonstrated the feasibility of preparing microemulsions with the highest content of protein and their long-term stability.

Evaluation of thermosensitive poloxamer 407 gel systems for the sustained release of estradiol in a fish model.

The purpose of this study was to develop and evaluate a delivery system comprising a thermosensitive gel for the sustained release of steroidal hormones in fish, over an extended period of time after a single intramuscular (i.m.) injection and for the improved reproductive performance in fish. Controlled delivery systems based on thermosensitive gels are easy to prepare, low cost and high versatility dosage forms, which have been shown to be effective in several animal species for sustained release of hormones. In this work, a thermosensitive gel system based on poloxamer 407 in water:ethanol medium, able to work as a prolonged release carrier for 17ß-estradiol (E2), has been developed. Such a system was able to solubilize the lipophilic E2 and to gel at the required water temperature for fish rearing (20°C). Moreover, the system exhibited the best injection condition at temperatures below 15°C when the system behaved as a low viscosity Newtonian liquid. The thermosensitive gel system was tested in vivo in the fish model, Carassius auratus, and the results compared with a single i.m. injection of E2 dissolved in corn-oil and other relevant control systems not containing E2. The results were particularly interesting, since fish injected with the E2 thermosensitive gel formulation, showed significantly higher levels of the circulating hormone than corn oil-E2 treated animals at 72 and 96h after injection. In addition, the thermogel system was able to sustain the plasma level of E2 for about 11days. The increased plasma levels of E2 were also accompanied by maintained higher values of plasma vitellogenin (VTG), thus suggesting that the thermosensitive polymer based delivery system could prevent rapid hepatic clearance of E2, resulting in prolonged stimulation of estrogen receptor-mediated pathways in goldfish.

A study on the stability and enzymatic activity of yeast alcohol dehydrogenase in presence of the self-assembling block copolymer Poloxamer 407.

Yeast alcohol dehydrogenase (ADH) is an enzyme widely studied for biotechnological applications due to its involvement in fermentation industry, and various attempts to improve its catalytic properties and its thermal stability have been carried out. In this paper, the influence of a block copolymer (Poloxamer 407) on ADH enzymatic activity and thermal behaviour has been studied in order to get new insights about the use of poloxamers in formulation of sustained release systems for therapeutic proteins. Poloxamer 407 has the ability to form micelles and gel due to its self-assembling and thermoresponsive properties. The effect of the copolymer towards thermal stress and pH changes, which often reduce enzymes activity it has been investigated by means of enzymatic assays and differential scanning calorimetry. Results showed that at pH 9.1 and 7.3, the Poloxamer in the form of unimeric, micellar and gel state is able to effectively preserve the enzyme from thermoinactivation. In addition by calorimetric data Poloxamer 407 has showed an effect in preserving ADH from aggregation at pH 7.3. In conclusion, Poloxamer 407 seems to be very effective in protecting ADH from stress related events, like alkaline inactivation and aggregation.

Monitoring the aggregation behaviour of self-assembling polymers through high-resolution ultrasonic spectroscopy.

Poloxamer 407 is a well-known self-assembling polymer with a wide range of temperature- and concentration-dependent phase behaviour, such as micellization and gelation. This work was carried out to demonstrate the potential of high-resolution ultrasonic spectroscopy in evaluating aggregation-deaggregation behaviour of self-assembling polymers. In order to achieve this objective, six different concentrations of Poloxamer 407 water dispersion were prepared and analysed between 5 and 35 degrees C using ultrasonic spectroscopy. For comparison, the same samples were also analysed by the DSC technique. The results showed that polymer aggregation process can be successfully monitored using both ultrasonic parameters of sound speed and attenuation. Furthermore, good agreement with DSC data was observed in terms of characteristic transition temperatures and also in terms of micellization kinetics and related parameters.