Controlled local release of PPARγ agonists from biomaterials to treat peripheral nerve injury.

OBJECTIVE: Poor clinical outcomes following peripheral nerve injury (PNI) are partly attributable to the limited rate of neuronal regeneration. Despite numerous potential drug candidates demonstrating positive effects on nerve regeneration rate in preclinical models, no drugs are routinely used to improve restoration of function in clinical practice. A key challenge associated with clinical adoption of drug treatments in nerve injured patients is the requirement for sustained administration of doses associated with undesirable systemic sideeffects. Local controlled-release drug delivery systems could potentially address this challenge, particularly through the use of biomaterials that can be implanted at the repair site during the microsurgical repair procedure. APPROACH: In order to test this concept, this study used various biomaterials to deliver ibuprofen sodium or sulindac sulfide locally in a controlled manner in a rat sciatic nerve injury model. Following characterisation of release parameters in vitro, ethylene vinyl acetate tubes or polylactic-co-glycolic acid wraps, loaded with ibuprofen sodium or sulindac sulfide, were placed around directly-repaired nerve transection or nerve crush injuries in rats. MAIN RESULTS: Ibuprofen sodium, but not sulindac sulfide caused an increase in neurites in distal nerve segments and improvements in functional recovery in comparison to controls with no drug treatment. SIGNIFICANCE: This study showed for the first time that local delivery of ibuprofen sodium using biomaterials improves neurite growth and functional recovery following PNI and provides the basis for future development of drug-loaded biomaterials suitable for clinical translation.

A spectroscopic and thermal investigation into the relationship between composition, secondary structure and physical characteristics of electrospun zein nanofibers.

Electrospun zein nanofibers have attracted interest as drug delivery systems due to their propensity for controlled drug release, flexible structure and low toxicity. However, comparatively little is known regarding the relationship between production method and fiber characteristics, both in terms of fiber architecture and protein structure. Here we use a range of imaging and spectroscopic techniques to elucidate the effects of solvent composition on zein secondary structure, fiber diameter and fiber integrity, plus we utilize the new technique of transition temperature microscopy to examine the thermal properties of the fibers. Zein nanofibers were prepared using ethanol, acetic acid and water mixes as solvents, alone and with plasticizers (polyethylene glycol, glycerol) and casein. Electrospinning was performed under controlled conditions and the products characterized using scanning electron microscopy (SEM), attenuated total reflection Fourier Transform infrared spectrometry (ATR - FTIR) and transition temperature microscopy (TTM). The choice of solvent, concentration and voltage, alongside the presence of additives (plasticizers and casein) were noted to influence both the diameter of the fibers and the tendency for bead formation. A relationship was noted between protein secondary structure and fiber architecture, with an enhanced ß-sheet content, enhanced by the inclusion of casein, being associated with higher beading. In addition, thermal imaging of electrospun zein fiber mats was successfully achieved using TTM via two dimensional mapping of the softening temperatures across the spun samples, in particular demonstrating the plasticizing effects of the polyethylene glycol and glycerol.

Humoral immune response against epidermal growth factor encapsulated in dehydration rehydration vesicles of different phospholipid composition.

We investigated the influence of dehydration-rehydration vesicles (DRV) phospholipid composition and the addition of other components on human recombinant epidermal growth factor (hrEGF) encapsulation efficiency and its release from liposomes. Encapsulation of EGF into DRV composed of phosphatidylcholine with different unsaturation levels was around 20-35%. The best result was obtained with dipalmitoyl phosphatidylcholine: cholesterol (DPPC:Ch) liposomes (35%) corresponding to the lowest hrEGF release during one month of storage. Even with this phospholipid composition, modification of the DRV procedure by including an extrusion step did not improve hrEGF encapsulation efficiency, rendering less stable particles. The inclusion of recombinant P64k from Neisseria meningitidis (rP64k), as such or conjugated to hrEGF, decreased the encapsulation efficiency of the latter protein into DRV or freeze and thaw multilamellar vesicles (FATMLV). The hrEGF release from liposomes could be related to the interaction between this polypeptide and the bilayer, as evidenced by increased carboxyfluorescein release from hrEGF-DRV; less susceptibility to fluorescence quenching by acrylamide in the presence of liposomes; and a measurable decrease of phospholipid phase transition Delta enthalpy (DeltaH). DRV comprising saturated phospholipids (DPPC:Ch or distearoyl phosphatidylcholine [DSPC]:Ch) and containing the conjugate EGF-P64k induced a more efficient immune response against hrEGF than unsaturated phospholipid and alum in terms of total IgG, IgG(2a), and IgG(2b) subclasses and the ability of antibody to inhibit the interaction of the EGF receptor with hrEGF.

The development of microthermal analysis and photothermal microspectroscopy as novel approaches to drug-excipient compatibility studies.

The use of microthermal analysis as a novel means of assessing chemical incompatibility between drugs and excipients is assessed using magnesium stearate and acetylsalicylic acid as a model system. Localised thermomechanical analysis (L-TMA), localised differential thermal analysis (L-DTA), nanosampling, thermally assisted particle manipulation (TAPM) and photothermal microspectrometry (PTMS) are developed as a means of allowing extremely small quantities of drug and excipient to be heated in close proximity to each other. Differential scanning calorimetry (DSC), hot stage microscopy (HSM) and temperature controlled attenuated total internal reflection (ATR) FTIR were used as supportive techniques. L-TMA and macroscopic TMA of magnesium stearate indicated that the endothermic DSC peak normally associated with melting does not correspond to significant liquefaction. An optimised method for detecting the interaction at a particulate level of scrutiny was developed whereby the drug is placed on the excipient surface via TAPM and the construct heated, allowing the interaction to be detected in both the L-TMA and L-DTA signal. PTMS allowed spectra to be obtained on nanogram-sized samples and also allowed the interaction to be detected. The study has therefore demonstrated the potential for using TAPM with PTMS for studying interactions at an individual particle level.

High sensitivity differential scanning calorimetry investigation of the interaction between liposomes, lactate dehydrogenase and tyrosinase.

High sensitivity differential scanning calorimetry (HSDSC) has been used to study the interaction of the model proteins lactate dehydrogenase (LDH) and tyrosinase with dimyristoylphosphatidylcholine (DMPC) liposomes, and relate this to the thermal and physical stability of the proteins. On heating, both LDH and tyrosinase denatured irreversibly in a time-dependent manner and modified the phase transition behaviour of DMPC liposomes at all concentrations investigated. The most marked effects occurred for the pretransition rather than the main phospholipid phase transition. The effects on the bilayer are likely to result from electrostatic interactions of the hydrophilic proteins with the head-groups of DMPC molecules, whilst due to their hydrophilic nature they do not penetrate into the bilayer. Tyrosinase is more highly ionised than LDH at the pH of the investigation, which may explain why tyrosinase has a greater effect than LDH on the HSDSC scans at mg/ml protein concentrations.

An investigation into the thermal behaviour of a model drug mixture with amorphous trehalose.

The thermal and structural properties of amorphous trehalose mixed with a model drug, paracetamol, have been studied with a view to developing understanding of the thermal events undergone by such binary systems. A physical mixture of paracetamol and spray dried trehalose (1:9 weight ratio) was studied using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), hot stage microscopy (HSM), and variable temperature powder X-ray diffraction (VTPXRD). The presence of the drug resulted in a lower temperature recrystallization exotherm for the trehalose compared to the disaccharide alone. Evidence is presented for the trehalose recrystallization being triggered by the melting rather than simply the presence of the paracetamol particles. HSM studies indicated that the trehalose recrystallized around the drug on heating, with the recrystallization temperature again corresponding to the melting of the drug. VTPXRD indicated that the presence of the drug again lowered the recrystallization temperature of the trehalose, although the trehalose anhydrate diffraction peaks were discernible at a lower temperature for both the pure trehalose and the mixed systems than was observed for the DSC studies, suggesting that the association between recrystallization and drug melting was not apparent when using this approach. It is suggested that while the trehalose recrystallization process is not significantly influenced by the presence of the drug when studied over relatively long time periods such as those used for the VTPXRD studies, the process is triggered by the melting of the paracetamol when short experimental times and scanning conditions are used such as those relevant to DSC studies. These data have implications for the quality control of trehalose products using DSC, the characterisation of the physical structure of the binary systems and the prediction of the corresponding physical stability.

Feasibility of a bioadhesive drug delivery system targeted to oesophageal tissue.

This contribution examines the feasibility of utilising an oesophageal-adhesive alginate layer to support model drug particles. Such a bioadhesive system offers the prospect of local drug delivery to the oesophagus, which in turn has applications in the treatment of conditions including gastro-oesophageal reflux disease and oesophageal cancer. Surface-modified (amine, carboxylate and sulfate) as well as neutral fluorescent beads were investigated as model drug particles. A fluorescence assay technique was utilised to quantify the extent and duration of adhesion of a fixed dose of these particles to excised porcine oesophageal tissue. Retention of the particles was investigated both from aqueous systems and within an adhesive alginate solution. After 30 min significantly higher adhesion of neutral beads was recorded from the alginate solution as compared to the aqueous suspension (n = 6, P < 0.05). The beads that possessed a negative charge showed significantly greater retention within the alginate carrier (n = 6, P < 0.05). However, the amine-modified beads showed retention profiles that were similar both within the alginate carrier and within the aqueous suspension (n = 6, P > 0.05).

An investigation into the structure and bioavailability of alpha-tocopherol dispersions in Gelucire 44/14.

In this investigation we describe the preparation, physical characterisation and in vivo behaviour of solid dispersions of a liquid nutraceutical, alpha-tocopherol, in Gelucire 44/14 with a view to establishing whether dispersion in this matrix may provide a means of formulating a liquid drug in a solid dosage form while also improving the oral bioavailability. Using Vitamin E Preparation USP as the source of alpha-tocopherol, dispersions were prepared using a melt-fusion method with active loadings up to 50% (w/w) and characterised using differential scanning calorimetry and optical microscopy. Capsules containing 300 IU alpha-tocopherol were manufactured and the absorption profiles compared to a commercial soft gelatin capsule preparation in healthy human volunteers. Confocal laser scanning microscopy (CLSM) studies were performed in order to elucidate the mechanism by which drug release may be occurring. Differential scanning calorimetry studies indicated that the presence of the active had a negligible effect on the melting profile of the carrier, indicating limited miscibility between the two components, a conclusion supported by the microscopy studies. Similarly, the dispersions were shown to exhibit a glass transition corresponding to the incorporated drug, indicating molecular cooperativity and hence phase separation from the lipid base. Despite the phase separation, it was noted that capsules stored for 18 months under ambient conditions showed no evidence of leakage. Bioavailability studies in six healthy male volunteers indicated that the Gelucire 44/14 formulation showed an approximately two-fold increase in total alpha-tocopherol absorption compared to the commercial preparation. Confocal laser scanning microscopy studies indicated that, on contact with water, the dispersions formed two interfacial layers, from which the Gelucire 44/14 disperses in the liquid medium as small particles. Furthermore, evidence was obtained for the dispersed material becoming incorporated into the hydrated lipid. In conclusion, the dispersion of the liquid drug in Gelucire 44/14 appears to allow the dual advantages of the preparation of a solid formulation and improved bioavailability of this material.

Characterization of ciclosporin A loaded poly (D,L lactide-co-glycolide) microspheres using modulated temperature differential scanning calorimetry.

The aim of this study was to investigate the physical structure of poly (D,L lactide-co-glycolide) (PLGA) microspheres loaded with ciclosporin A in terms of the amorphous properties of the individual components and the phase separation characteristics of the binary systems. Microspheres were prepared using a standard oil-in-water emulsion technique. The thermal properties of the PLGA, ciclosporin A and loaded spheres were investigated using modulated temperature differential scanning calorimetry (MTDSC) using a TA Instruments MTDSC 2920, with scanning electron microscopy (SEM), X-ray powder diffraction (XRD) and high-performance liquid chromatography used as supportive techniques. MTDSC indicated a glass transition for ciclosporin A in the reversing heat flow signal at 107 degrees C, supported by temperature cycling studies, while XRD showed clear evidence for diffraction peaks, thereby indicating that the material as received is semi-crystalline. The unloaded PLGA spheres showed a glass transition (Tg) at 43 degrees C, with no reduction in Tg being observed on loading the peptide up to 50%, w/w. Similarly, no evidence for diffraction peaks were seen for the drug-loaded systems, although the glass transition corresponding to the peptide was observed for the loaded microspheres, suggesting that the drug is present as a separate amorphous phase. Similarly, SEM studies showed the appearance of distinct "islands" on the surface of the spheres that are suggested to correspond to the drug phase, with the size of the islands increasing with drug loading. Evidence is therefore presented that ciclosporin A may exist in a range of solid states, with the degree of crystallinity being altered by processing. In addition, there appears to be little or no miscibility between the drug and PLGA using the manufacturing protocol employed here. These findings may have implications for the choice of manufacturing protocol, the release of peptide drugs from PLGA microspheres and the chemical and physical stability of such drugs.

Characterisation of water behaviour in cellulose ether polymers using low frequency dielectric spectroscopy.

The behaviour of water in hydroxypropylmethylcellulose (HPMC) K100LV, K4M, K15M, K100M, E4M, F4M and HPC polymers was characterised using low frequency dielectric spectroscopy (LFDS). Dielectric responses of 25% (w/w) HPMC K15M gels and deionised water were found to be similar at +22 and 0 degrees C. However, at -30 degrees C, a dielectric response typical of a solid was apparent. The melting of frozen water within gels was detected as increases in the magnitude of the dielectric response with increase in temperature. More than one phase transition was visible in the majority of gels studied which may be related to the presence of different states of water melting at different temperatures. In addition to polymer concentration, both polymer molecular weight and substitution level influenced the nature of the transitions. The magnitude of the dielectric response was increased in all HPMC gel systems in comparison to the response seen in deionised water. Drug addition affected the transitions occurring during the melting of ice in the gels. This may be related to the presence of ionic species in the systems. LFDS studies on cellulose ether gels have provided some interesting evidence for the existence of more than one state of water within such gel systems. The results are in good agreement with thermal analysis findings in similar gel systems.

An in vitro mucosal model for prediction of the bioadhesion of alginate solutions to the oesophagus.

This paper discusses the development of an in vitro model utilised to assess the adhesion of alginate solutions to porcine oesophageal tissue. The methodology involved the construction of retention apparatus onto which sections of tissue were mounted. Fluorescently labelled alginate solutions of known rheological profile were dispensed onto the tissue at a concentration of 2% w/v. A washing solution was applied at a specified rate to mimic saliva flow and the eluted material collected. Fluorimetric analysis allowed dose retention to be assessed as a function of time. The effect of the nature of the washing solution and the choice of alginate were investigated. It was found that after 30 min up to 20% of the applied alginate dose remained associated with the tissue, regardless of the alginate selected from the range examined. The nature of the washing medium did not have a significant effect on retention, irrespective of the inherent mucin concentration. Overall this study indicated that the technique presented offers a viable means of studying bioadhesion of liquids and also demonstrates that alginate solutions may have an application as bioadhesive agents for localisation within the oesophagus.

Characterization of amorphous ketoconazole using modulated temperature differential scanning calorimetry.

The objective of the present study was to characterize the glassy state of ketoconazole and to calculate its molecular mobility below the glass transition, with a view to further developing the use of modulated temperature differential scanning calorimetry (MTDSC) as a means of studying relaxation behavior. Particular emphasis is placed on identifying the influence of the choice of experimental parameters on the measured values of both the glass transition temperature (T(g)) and the relaxation enthalpy magnitude. Amorphous ketoconazole was studied using an amplitude of +/-0.212 K, a period of 40 s, and an underlying heating rate of 2 K/min. The correction required for the calculation of the relaxation endotherm magnitude (the "T(g) shift effect") was demonstrated and is discussed in terms of the mechanism underpinning this phenomenon. Similarly, the influence of the choice of MTDSC experimental parameters on the measured T(g) was studied by varying the amplitude from +/-0.011 to +/-0.424 K and the period from 25 to 50 s. The influence of the cooling rate from the melt on the magnitude of the relaxation endotherm and position of the glass transition was investigated. It was noted that the magnitude of the relaxation endotherm increased with slower cooling rates, this being ascribed to a combination of annealing during the cooling and heating cycle and a further facet of the T(g) shift effect. Annealing experiments were performed at aging temperatures T(g)-12--T(g)-42 K for periods ranging from 10 min up to 16 h. The relaxation behavior was characterized by fitting the calculated extent of relaxation to the Williams-Watts equation. Overall, the study has highlighted theoretical and experimental issues that need to be considered when using both DSC and MTDSC for the calculation of relaxation times.

An assessment of jet and ultrasonic nebulisers for the delivery of lactate dehydrogenase solutions.

The aim of this study was to investigate the suitability of commercial jet and ultrasonic nebulisers for effective delivery of the model hydrophilic protein lactate dehydrogenase (LDH). Two jet nebulisers (Pari LC Plus and Pari LC Star) and two ultrasonic nebulisers (Sonix 2000 and Omron U1) were used to nebulise LDH solutions and the effects on protein activity and protein concentration determined. The size distribution of the aerosols produced, measured by laser diffraction analysis, temperature changes during nebulisation, the time to atomise a 5 ml dose volume and the mass output of the four nebulisers were compared. A twin impinger (TI) was used to collect the nebulised protein, which was assayed for total and active protein content. There was a large variation in the median size and size distribution of the aerosols produced by each of the nebulisers from LDH and Sørensen's modified phosphate buffer, and in the time taken to reach the sputtering phase of aerosolisation. During use, the concentration of LDH increased in the Omron U1 nebuliser, but did not change significantly in the others. The temperature of the protein solution decreased by approximately 8 degrees C during jet nebulisation but increased by 3 and 10 degrees C in the Omron U1 and Sonix 2000 nebulisers, respectively. Denaturation of LDH within the nebuliser reservoir, occurred in the order Sonix>Pari LC Plus>Pari LC Star>Omron U1, whilst the deposition of active and total protein within the stages and throat of the TI was a function of the particle size of the aerosols generated and the specific device used.

An investigation into the effects of residual water on the glass transition temperature of polylactide microspheres using modulated temperature DSC.

The objective of the study was to ascertain residual water levels in polylactide and polylactide-co-glycolide microspheres prepared using the solvent evaporation technique and to investigate the effects of that water on the glass transitional behaviour of the microspheres. Microspheres were prepared from polylactic acid (PLA) and polylactide-co-glycolide (PLGA) 50:50 and 75:25 using a standard solvent evaporation technique. The glass transition was measured as a function of drying conditions using modulated temperature DSC. The microspheres were found to contain very low levels of dichloromethane, while residual water levels of up to circa 3% w/w were noted after freeze or oven drying, these levels being higher for microspheres containing higher glycolic acid levels. The residual water was found to lower the T(g) following the Gordon-Taylor relationship. The data indicate that the microparticles may retain significant water levels following standard preparation and drying protocols and that this drying may markedly lower the T(g) of the spheres.

An investigation into the thermal behaviour of an amorphous drug using low frequency dielectric spectroscopy and modulated temperature differential scanning calorimetry.

The objective of the study was to investigate the use of low frequency dielectric spectroscopy as a means of characterizing the thermal transitions of an amorphous drug substance, indometacin, with particular emphasis on modelling the response using the Dissado-Hill function. The low frequency dielectric behaviour of indometacin was measured over a temperature range of 10-160 degrees C and a frequency range of 10(-3)-10(6) Hz. Modulated temperature differential scanning calorimetry (MTDSC) studies were also performed on equivalent samples, showing a glass transition, recrystallization and melting. Isothermal low frequency dielectric spectra of the sample at temperatures below recrystallization showed the dynamic dielectric relaxation associated with the amorphous phase, while changes in the real and imaginary permittivities were observed that were associated with recrystallization and subsequent melting. A small discontinuity was observed immediately above the recrystallization process in the MTDSC and dielectric data, suggested to correspond to a solid state transformation. The use of the Dissado-Hill function as a means of modelling the dielectric behaviour has also been described. The study suggests that low frequency dielectric spectroscopy, used in conjunction with MTDSC and Dissado-Hill modelling, may be a useful tool for the characterization of amorphous and crystalline drugs.

The measurement of beclomethasone dipropionate entrapment in liposomes: a comparison of a microscope and an HPLC method.

The purpose of this study was to examine the methodologies that may be used to estimate the maximum incorporation (<5 mole% drug) of beclomethasone dipropionate (BDP) in dipalmitoylphosphatidylcholine (DPPC) multilamellar liposomes. Two approaches are described. First, differential interference contrast (DIC) microscopy and cross-polarisation microscopy have been used to measure the concentration at which BDP crystals become apparent in BDP-containing liposome preparations, thereby allowing a semi-quantitative but simple estimation of entrapment. An alternative method is described whereby the unentrapped solid drug is separated from the liposomes via suspension in D2O, followed by centrifugation and HPLC analysis. The method resulted in an estimate of 1.5-2 mole% BDP, while the HPLC method yielded a value of 2.52 mole% BDP.

Low frequency dielectric investigations into the relaxation behavior of frozen polyvinylpyrrolidone-water systems.

The low frequency dielectric response of aqueous solutions containing 0, 1, 5, and 10% w/v polyvinylpyrrolidone (PVP) was studied to characterize the low temperature relaxation behavior of these systems. Complementary modulated temperature differential scanning calorimetry (MTDSC) studies allowed measurement of the glass transition temperature for these materials, corresponding to the behavior of the nonfrozen phase. Dielectric investigations in the frequency range of 10(6) to 10(-2) Hz were performed on the systems in the liquid state, with a Maxwell-Wagner response noted for both the PVP solutions and water. The solid-phase responses were studied over a range of temperatures down to -70 degrees C, with a relaxation peak observed for the PVP systems in the kilohertz region. The spectra were modeled using the Havriliak-Negami equation and the corresponding relaxation times were calculated, with a satisfactory fit to the Arrhenius equation noted. The calculated activation energies were similar to literature values for the dielectric relaxation of water. It is suggested that the dielectric response is primarily a reflection of the relaxation behavior of the water molecules in the nonfrozen fraction, thereby indicating that the dielectric technique may yield insights into specific components of frozen aqueous systems.

An investigation into the low temperature thermal behaviour of vitamin E preparation USP using differential scanning calorimetry and low frequency dielectric analysis.

The thermal and dielectric responses of Vitamin E Preparation USP have been examined to further understand the melting and solidification of this material. A TA Instruments 2920 Differential Scanning Calorimeter was used to examine the thermal response of the sample at a range of scanning speeds. Isothermal dielectric studies were performed using a Novocontrol Dielectric Spectrometer over a range of temperatures down to -70 degrees C and a frequency range of 10(6)-10(-2) Hz. The differential scanning calorimetry (DSC) studies showed an anomalous response whereby at slow heating rates (2 degrees C min(-1)) a small exotherm followed immediately by an endotherm was observed. This response was considerably diminished in magnitude at higher rates (5 degrees C min(-1)) and was not observed at the fastest heating rate of 10 degrees C min(-1). No thermal events were seen on cooling the sample to -60 degrees C. It was suggested that the material formed a glass on cooling, with a predicted transition temperature of approximately -100 degrees C. Further studies using a liquid nitrogen cooling system indicated that the system did indeed exhibit a glass transition, albeit at a higher temperature than predicted (ca -63 degrees C). Low frequency dielectric analysis showed a clear relaxation peak in the loss component, from which the relaxation time could be calculated using the Havriliak-Negami model. The relationship between the relaxation time and the temperature was studied and was found to follow the Vogel-Tammann-Fulcher (VTF) modification of the Arrhenius equation. It is therefore concluded that Vitamin E Preparation USP is a glass-forming material that exhibits kinetically-hindered recrystallisation and melting behaviour. The study has also indicated that DSC and low frequency dielectric analysis may be powerful complementary tools in the study of the low temperature behaviour of pharmaceuticals.