Controlled drug delivery to the lung: Influence of hyaluronic acid solution conformation on its adsorption to hydrophobic drug particles.

This work reports investigations into the interaction and adsorption of the hydrophilic polymer hyaluronic acid (HA) onto the surface of the hydrophobic corticosteroid drug fluticasone propionate (FP). The eventual aim is to formulate a bioadhesive pulmonary drug delivery system with prolonged action that avoids rapid clearance from the lungs by the mucociliary escalator. Adsorption isotherms detailing the adsorption of HA from aqueous HA solution concentrations ranging from 0.14 to 0.0008% (w/v) to a fixed FP particle concentration of 0.1% (w/v) were investigated. The method of preparing FP particles with HA molecules adsorbed on their surfaces (FP/HA particles) involved suspension of the FP either in hydrated HA solution or in water followed by addition of solid HA, centrifugation of the solids to form a pellet, washing the pellet several times with water until no HA was found in the supernatant and then freeze drying the suspension obtained by dispersing the final pellet. The freeze dried powder was then analysed for adsorbed HA using a Stains-all assay. The influence of order of addition of HA to FP, time for the adsorption process, and temperature of preparation on the adsorption isotherms was investigated. The non-equilibrium adsorption isotherms produced generally followed the same trend, in that as the HA solution concentration increased, the amount of HA adsorbed increased to a maximum at a solution concentration of approximately 0.1% (w/v) and then decreased. The maxima in the adsorption isotherms were close to the change from secondary to tertiary conformation in the HA solutions. Below the maxima, adsorption occurred via interaction of FP with the hydrophobic patches along the HA chains in the secondary structures. Above the maxima, secondary HA molecules aggregate in solution to form tertiary network structures. Adsorption from tertiary structure was reduced because strong interactions between the HA molecules limited the availability of hydrophobic patches for adsorption of HA onto FP. The influence of preparation variables on adsorption was also related to the availability of hydrophobic patches for adsorption.

Adsorption of enzymes at the solid-liquid interface. I. Trypsin on polystyrene latex.

The enzyme, trypsin, has been used to study conformational changes which occur when protein adsorption onto well-characterized, emulsifier-free, polystyrene latex surface takes place. The adsorption isotherm is of the high affinity, Langmuirian type with plateau adsorption of trypsin of 2.8 mg m-2. The enzymic activity of adsorbed trypsin to low molecular weight substrate is found to decrease as the surface coverage decreases indicating that 'spreading' or unfolding of the native protein conformation, with consequent loss of enzymic activity, occurs. On the close packed surface such 'spreading' is inhibited by steric factors. The view that protein adsorption onto hydrophobic surfaces is dominated by the entropy gain due to protein unfolding to maximize hydrophobic interactions is thus supported.

Incorporation and release of chemically intact mitomycin C from albumin microspheres: a high performance liquid chromatography evaluation.

Preparation of mitomycin C-loaded human serum albumin (HSA) microspheres using a new technique that avoids the use of heat denaturation, which is known chemically to degrade incorporated drug, is described. This method is based on cross-linking of protein by glutaraldehyde (2.2%) during emulsification (W/O) at room temperature. The resultant particles have a mean (s.d.) diameter of 16.9 (0.34) microns (50% weight average), contain mean (s.d.) 1.15 (0.05%) mitomycin C (MMC) (w/w, n = 17) and maintain sustained release of drug over 20 h. High performance liquid chromatography (HPLC) with diode array detection was used to study the chemical integrity of the drug. Two classes of decomposition products were evaluated: chemical degradation products and drug/nucleophile covalent adducts. The HPLC separation was validated by a number of standards of proposed degradation products. To examine incorporated drug, a complete microsphere system was solubilized with 0.4% trypsin for 24 h, while to examine released drug, microspheres were immobilized on a flow-through glass wool column and fractions were collected. No evidence of significant chemical degradation or covalent coupling to protein was detected in microsphere digests. Two candidate decomposition products, representing approximately 10% of drug released from microspheres (assuming similar molar extinction coefficients to MMC), were identified in column fractions. One of these products appeared to be a covalent adduct, the other possibly an isomeric form of intact MMC. Thus, MMC is predominantly incorporated into and released (90%) chemically intact from HSA microspheres prepared by the technique described.

Investigation of factors affecting adhesion of 99Tcm labelled colloids to glass vials.

Factors which may possibly influence the adhesion of 99Tcm labelled colloid to glass vials were investigated. Of five colloids studied, two protein-free tin colloids were affected most by the problem, some 12-18% being lost by adhesion to the glass vial when the colloid was left resting on the bench, or 60-87% when it was rotated for a period of 4 h. The proportion of activity which adhered to the vial was dependent on the time the product had been contained within it, whether on the bench or rotated. Two of the colloids, both of which contained protein, were affected only slightly by the problem, one being a tin colloid and the other a sulphur colloid. An antimony sulphide colloid, which contained no added protein, was virtually unaffected by the problem. The degree of adhesion was not related to the pH of the product, nor to the temperature at which the product was stored, nor to the tendency of the colloid particles to grow in size upon vigorous agitation.

Formation of large particles in a 99Tcm-tin colloid preparation.

The effect of prolonged continuous movement on particle size within a 99Tcm-tin colloid preparation was investigated, making use of photon correlation spectroscopy for size estimation. No increase in particle size was found when the preparation was left undisturbed for up to 7 h, the mean (+/- S.D.) size being found to be 239 +/- 24 nm. When the preparation was placed in a laboratory rotator for the same period of time, there was a gradual increase in mean particle size to 404 +/- 127 nm. The increase in size was much greater, however, after 7 h of continuous agitation in a laboratory shaker, a mean value of 2260 +/- 746 nm being observed. An antimony sulphide colloid was subjected to continuous agitation for the same period of time but there was no evidence of aggregation. It is supposed that continuous movement of reconstituted 99Tcm-tin colloid, leading to a marked increase in particle size, may explain occasional instances of significant lung intake.

Formation of labelled colloid in 99Tcm-DMSA due to the presence of bactericidal fluid.

The possibility that traces of bactericidal fluid in 99Tcm-DMSA could lead to the formation of labelled colloid, was explored. In vitro investigations were undertaken using ultracentrifugation techniques and photon correlation spectroscopy. The latter showed that both contaminated and uncontaminated DMSA contained colloidal (or particulate) material. However the presence of 10 microliters bactericidal fluid as contaminant was shown by ultracentrifugation to result in labelling of this colloidal material when 99Tcm was added to DMSA. Studies in a normal volunteer confirmed the results of the in vitro studies, in that significant liver and spleen uptake was observed after the administration of contaminated 99Tcm-DMSA.

Immunoscintigraphy of human ovarian cancer xenografts using a radiolabelled monoclonal antibody to human pregnancy serum-derived immune complexes.

The in vivo imaging of xenografted human ovarian cancer in nude mice with a specific and control radiolabelled monoclonal antibody (MoAb) is described. The specific MoAb was previously raised by immunizing mice with immune complexes derived from late human pregnancy serum. In the first group of mice the specific MoAb 131I-5E3 F(ab')2 was injected, while a second group received equivalent amounts of a control MoAb 131I-UJ13A F(ab')2. The mice were imaged at various times up to a maximum of 2 weeks using a gamma camera, and the tumour to non-tumour (T/NT) ratio was recorded for each group. The T/NT ratio rose to 2.02 in the specific group, while the corresponding ratio in the control group was 0.70. In addition, the count rate in the tumour and non-tumour regions was determined on each imaging occasion. Biological half-lives of the divalent fragments of 5E3 and UJ13A in the tumour were 7.53 days and 0.62 days, respectively. Following sacrifice, the tumours were excised and counted relative to the rest of the animal, and the T/NT ratio was calculated. In vitro results were in direct agreement with those recorded in vivo using the gamma camera. From the results it would appear that the divalent fragment of 5E3, which has been raised to immune complexes derived from late human pregnancy serum, is specific for human ovarian tumour xenografts in the nude mouse model.

A study of enzyme and protein microencapsulation--some factors affecting the low apparent enzymic activity yields.

Microencapsulation of aqueous solutions of enzymes, alpha-chymotrypsin (EC 3.4.21.1) and histidase (EC 4.3.1.3), with semipermeable polyamide membranes resulted in a loss of enzymic activity. The low yields (less than 40%) found with both enzymes were typical of others reported in the literature. The activity of broken histidase-containing microcapsules was greater than that of the microcapsules before breaking, and this was interpreted as being due to a simple diffusional restriction on the substrate and product. The maximum of the apparent pH-activity curve of alpha-chymotrypsin was found to be shifted one unit to more alkaline pH when the enzyme was encapsulated. This phenomenon was explained in terms of the hydrogen ion concentration in the microenvironment surrounding the enzyme being different from that in the bulk solution. Microencapsulation of aqueous solutions of enzymes is accomplished by in situ polymerization reactions at the interface of a water-in-oil emulsion. 125I-labelled proteins (albumin and fibrinogen) were encapsulated under similar conditions to determine the efficiency of the microencapsulation process. About one third of these proteins was lost during the overall preparation procedure and a further fraction was attached to the membranes of the microcapsules.

The effect of hydrotropic agents on the heat coagulation of bovine serum albumin.

The effect of two hydrotrophic solubilizers on the heat coagulation of bovine serum albumin (BSA) has been investigated. Photon correlation spectroscopy indicated possible unfolding of BSA molecules in solutions of sodium benzoate and sodium salicylate at 25 degrees C. The effect of these hydrotropes on the heat coagulation of BSA was concentration-dependent. Relatively low concentrations stabilized the protein structure as indicated by the increase in the transition temperature(Tm) and induced gelation at temperatures and BSA concentrations lower than those required in the absence of hydrotropes. Higher concentrations of the hydrotropes considerably reduced Tm and inhibited gelation of BSA, the effect of sodium salicylate being more pronounced, as was the lower aggregation rate of BSA. The behaviour of these hydrotropes as protein denaturants differs from that of neutral electrolytes but is similar to that of concentrated solutions of urea.

The nature of the oil phase and the release of solutes from multiple (w/o/w) emulsions.

The effect of the nature of the oil phase of w/o/w emulsions stabilized by interfacial complexation between span 80 (sorbitan mono-oleate) and albumin has been studied. The long-term stability of the systems has been assessed by photomicrography and by measuring the quantity of an internal marker (NaCl) remaining entrapped with time. The number of multiple oil drops and the diameters of the internal aqueous droplets were determined over 6 weeks, and the amounts of NaCl entrapped over the same period were followed. There were no significant changes in w/o/w emulsions prepared with a range of hydrocarbons (octane, dodecane, hexadecane, toluene and cyclohexane), indicating stable multiple emulsions. The release of NaCl and 5-fluorouracil (5-FU) separately entrapped in the internal aqueous phase of w/o/w emulsions was measured. Diffusion of the un-ionized species of 5-FU across the oil phase or through localized thin oil lamellae is the primary transport mechanism. In the presence of surface active agents, water is solubilized in inverse micelles which would possess the ability to solubilize other water-soluble components, such as NaCl and 5-FU. The mixed inverse micellar units of Span 80 and polysorbate (Tween) 80 therefore act as solute carriers across the liquid hydrocarbon membrane separating the two aqueous phases of the emulsions. The main factor in determining the differences in rates of release from the hydrocarbon emulsions appears to be the droplet size of the internal aqueous phase.

Polymeric biomaterials: influence of phosphorylcholine polar groups on protein adsorption and complement activation.

The introduction to polymeric biomaterials of phosphorylcholine polar groups represents an approach towards the development of materials with improved blood compatibility. In this respect, two biomaterials, one a copolymer of butyl methacrylate and 2-methacryloyloxyethylphosphorylcholine (MPC), (poly(BMA-co-MPC) and the other, MPC-grafted Cuprophan, were examined with respect to their influence on protein adsorption and complement activation. Protein adsorption was studied by measurement of the adsorption of radiolabelled single proteins (albumin and fibrinogen), while complement activation was measured using radioimmunoassay for C3a des Arg. The investigation demonstrated that the polymers containing phosphorylcholine polar groups can achieve a marked reduction in protein adsorption and complement activation and supports the utilization of phosphorylcholine polar groups as a means of improving the compatibility of biomaterials for blood-contacting applications.