A rheological assessment of the nature of interactions between mucoadhesive polymers and a homogenised mucus gel.

The ability of mucoadhesive materials to produce a large increase in the resistance to deformation when incorporated into a mucus gel, relative to when the mucus gel and test materials are evaluated separately at the same concentration, has been reported in several previous studies. It has been proposed that this phenomenon, termed rheological synergism, can be used as a measure of the strength of the mucoadhesive interaction. This study investigated the interactions between four putative mucoadhesive polymers (Noveon, Pemulen TR-2, carageenan and sodium carboxymethylcellulose) and a homogenised mucus gel, using dynamic oscillatory rheology. It was shown that, with the exception of sodium carboxymethylcellulose, incorporating a mucoadhesive polymer into a mucus gel produces rheological behaviour indicative of a weakly cross-linked gel network, which suggested a structure containing physical chain entanglements and non-covalent (probably hydrogen) bonds. Optimum gel strengthening occurred in a weakly acidic environment, suggesting an optimum conformation and degree of ionisation of the polymer and mucus molecules. Subsequent work suggested that the macromolecular interactions between polymer and mucus are sensitive to temperature, with the dynamic moduli decreasing with increasing temperature, further indicating bonding of a non-covalent nature. This work provide further evidence that rheological methods can be used as a tool to evaluate the interactions between a mucoadhesive macromolecule and a mucus gel. It also adds to the perception that molecular interpenetration may be an important factor in mucoadhesion by strengthening the mucus in the mucoadhesive/mucosal interfacial layer.

The gastrointestinal transit profile of 14C-labelled poly(acrylic acids): an in vivo study.

The gastrointestinal distribution profiles for three 14C-labelled poly(acrylic acid)s of different average molecular weights and degrees of cross-linking have been established using the rat model. Despite initial differences in transit times and retention characteristics, these structural features were found to be of little influence to the overall gastrointestinal transit of the materials under consideration. No evidence for the systemic absorption of any of the polymers could be identified.

The mycobactericidal efficacy of ortho-phthalaldehyde and the comparative resistances of Mycobacterium bovis, Mycobacterium terrae, and Mycobacterium chelonae.

OBJECTIVES: To assess the mycobactericidal efficacy of an agent relatively new to disinfection, ortho-phthalaldehyde (OPA) and to compare the resistances of three Mycobacterium species. Mycobacterium bovis (strain BCG) was compared with Mycobacterium chelonae and Mycobacterium terrae to investigate the feasibility of using either of the latter two species in tuberculocidal testing. M. chelonae (a rapid grower) and M. terrae (an intermediate grower) both grow faster and are less virulent than M. bovis (a slow grower). DESIGN: The quantitative suspension protocol specified by the Environmental Protection Agency (EPA), the Tuberculocidal Activity Test Method (EPA test), was used throughout this study. Standard suspensions of all three species were prepared in a similar manner. Two suspensions of M. bovis, created in different laboratories, were used. These were tested against two concentrations of alkaline glutaraldehyde to provide reference data. Two concentrations of OPA were evaluated against all mycobacterial test suspensions. Four replicates of each organism-disinfectant combination were performed. RESULTS: Results were assessed by analysis of variance. M. terrae was significantly more resistant to 0.05% OPA than either M. bovis or M. chelonae. At 0.21% OPA, M. terrae was slightly more susceptible than one test suspension of M. bovis, but not significantly different from the other. M. chelonae was significantly less resistant than the other species at both OPA concentrations. At their respective minimum effective concentration, OPA achieved a 6-log10 reduction of M. bovis in nearly one sixth the time required by glutaraldehyde (5.5 minutes vs. 32 minutes). CONCLUSIONS: These data, along with other recent studies, lend support to the idea that M. terrae may be a suitable test organism for use in the tuberculocidal efficacy testing of disinfectants. They also confirm the relatively rapid tuberculocidal activity of OPA.

A rheological examination of the mucoadhesive/mucus interaction: the effect of mucoadhesive type and concentration.

The ability of mucoadhesive polymers to produce a large increase in the resistance to deformation when incorporated into a mucus gel, relative to when the mucus gel and test materials are evaluated separately at the same concentrations, has been reported in several previous studies. It has been proposed that this phenomenon, termed rheological synergism, can be used as a measure of the strength of the mucoadhesive interaction. In this investigation rheological synergism was investigated for a large range of putative mucoadhesive gels by dynamic oscillatory rheology. Changes in the storage modulus (G'), loss modulus (G"), and loss tangent (tan-delta) were found and the relative rheological synergism calculated. Rheological synergism was evident for a range of materials with known mucoadhesive properties giving behaviour between that of strongly cross-linked polymers gels and a physically entangled system. This effect was most marked with materials known to be of high mucoadhesive strength even when fully hydrated. Hence, polymers like sodium carboxymethylcellulose and high molecular weight poly(ethylene oxide) showed limited rheological synergism. When the effect of mucoadhesive concentration was investigated, relative rheological synergism occurred within a limited concentration range only, the values of which varied between materials. It was concluded that macromolecules possessing numerous hydrogen bond-forming groups and an open expanded network in the test environment gave pronounced rheological synergism, and the relevance of this to mucoadhesion studies will be investigated in further work. This study also confirmed the advantages of dynamic oscillatory rheology over simple viscosity measurement in the study of these systems.

Azocross-linked poly(acrylic acid) for colonic delivery and adhesion specificity: synthesis and characterisation.

A new synthetic pathway to 4,4'-divinylazobenzene is presented together with a procedure for the copolymerisation of this compound with acrylic acid. The chemical structure of the synthesised series of copolymers is examined in the light of infrared spectroscopy results and nuclear magnetic resonance data. The thermal properties of the materials are assessed using a combination of thermal analysis techniques and their swelling behaviour is evaluated at physiologically relevant buffers designed to mimic the gastrointestinal environment.

Azocrosslinked poly(acrylic acid) for colonic delivery and adhesion specificity: in vitro degradation and preliminary ex vivo bioadhesion studies.

This study reports on the performance of a novel polymeric material that is capable of providing site specificity in active agent delivery and the development of mucoadhesive interactions. Azo-networks, based on an acrylic backbone crosslinked with 4,4'-divinylazobenzene, were subjected to in vitro degradation and mucoadhesion (before and after degradation) testing in order to model their performance in the gastrointestinal tract. Advanced surface characterisation techniques (SEM, AFM, FTIR microscopy) were used to examine the network morphology prior to, and after degradation. The data obtained from these studies indicate that there is an optimum crosslinking density to allow non-adhesive particles to reach the colon. Within the colonic environment, the azo network degrades to produce a structure capable of developing mucoadhesive interactions with the colonic mucosa.

An in vitro mucosal model predictive of bioadhesive agents in the oral cavity.

The formulation of a drug/carrier complex that can be distributed and retained for extended periods within the oral cavity would be advantageous in the treatment of local conditions. In this study, an in vitro system was developed to investigate the binding of bioadhesive macromolecules to buccal epithelial cells, without having to alter their physicochemical properties by the addition of 'marker' entities. In this innovative approach a lectin binding inhibition technique, involving an avidin-biotin complex and a colourmetric detection system, was used to evaluate polymer binding. 0.5% w/v polymer solutions in saline (pH 7.6) were left in contact with a standardized number of freshly collected human buccal cells for 15 min. The cells were then exposed to 10 mg L(-1) biotinylated lectin from Canavalia ensiformis followed by 5 mg L(-1) streptavidin peroxidase. The inhibition of lectin binding (i.e. by 'masking' of the binding site on the cell surface by the attached bioadhesive polymer) was measured and expressed as a percentage reduction in the rate of o-phenylenediamine oxidation over 1 min. From the wide range of polymer solutions screened, chitosan gave the greatest inhibition of lectin binding to the surface of buccal cells, while methylcellulose, gelatin, Carbopol 934P and polycarbophil also produced a substantial reduction. Lectin binding inhibition was also observed for a selected number of polymer solutions when screened at pH 6.2. The presence of bound chitosan, polycarbophil and Carbopol 934P on the buccal cell surface was confirmed using direct staining techniques. It was concluded that this assay can be used to detect polymer binding to the cells present on the buccal mucosa, and the information gained used in the development of retentive drug/polymer formulations.

A direct-staining method to evaluate the mucoadhesion of polymers from aqueous dispersion.

A novel technique to evaluate polymer adhesion to human buccal cells following exposure to aqueous polymer dispersion, both in vitro and in vivo, is described. Adhering polymer has been visualised by staining with 0.1% (w/v) of either Alcian blue (60 min) or Eosin (10 min) solution, uncomplexed dye being removed by 0.25 M sucrose washings. The extent of polymer adhesion was quantified by measuring the relative staining intensity of control and polymer-treated cells by image analysis. In vitro, Carbopol 974P, polycarbophil (Noveon AA-1) and chitosan (CL 113) were found to adhere to human buccal cells from 0.10% (w/w) aqueous dispersions of these polymers. Following in vivo administration as a mouthwash, these polymers persisted upon the human buccal mucosa for at least 1 h.

Lectins for drug delivery within the oral cavity--investigation of lectin binding to oral mucosa.

The aim of this study was to identify receptors present on the buccal mucosa in order to select appropriate lectins that will allow the retention of a dosage form within the oral cavity. Studies using human buccal cells, the avidin-biotin-complex/diaminobenzidine method for identifying lectin binding and a microdensitometer to allow a semi-quantitative analysis of stain intensity, showed a wide diversity of lectin receptors. Kinetic studies of lectin binding to buccal cells revealed significant binding after 20 s, particularly for lectins from Pisum sativum and Arachis hypogaea. A significant reduction in lectin binding was observed after exposing buccal cells to a series of lectin solutions pre-treated with a large excess of secretor or non-secretor saliva. However when bound to the buccal cells, there was little displacement of lectins on exposure to either saliva types. Further studies on rat oral tissue suggested that the lectins appeared to bind to varying degrees on whole oral epithelial surfaces although differences in binding between this and the human buccal cell model were evident. It was concluded that a wide range of possible target receptors for lectins are present on rat oral epithelium and human buccal cells. Lectin binding to these receptors can be affected by the exposure time and the presence of saliva.

An in-vitro evaluation of lectin cytotoxicity using cell lines derived from the ocular surface.

Lectins are proteins or glycoproteins of non-immune origin capable of binding to one or more specific sugar residues. The potential for using lectins as a means of locating and "anchoring" a drug delivery system to a target site on a mucosal surface in order to promote controlled drug delivery and enhanced absorption has been described in previous work. Toxicity is evident for many lectins, and this study has investigated the cytotoxicity of lectins to cells derived from the cornea and conjunctiva. Cultures of transformed cell lines from the conjunctiva and cornea were exposed to solution of lectins, and the viability of these cells assessed by their ability to take up an MTT stain relative to the control. Clear evidence of lectin cytotoxicity was apparent for all of the lectins tested in this study, particularly at higher concentrations and over the longer (48 h) time period. This would raise issues with their potential use in targeted drug delivery systems. The lectins from Lycopersicon esculentum and Helix pomatia appeared to show the least cytotoxic activity and so could be considered the most promising. The lectins from Solanum tuberosum and Triticum vulgaris showed significant cytotoxicity, which contrasts with previous in-vivo studies. However, they might be considered for investigation as targeted therapeutic agents in the treatment of malignant diseases.

Lectins in drug delivery: a study of the acute local irritancy of the lectins from Solanum tuberosum and Helix pomatia.

Lectins are proteins or glycoproteins of non-immune origin capable of binding to one or more specific sugar residues. The potential for using lectins as a means of 'anchoring' a drug delivery system to the mucosal surfaces of the eye has been investigated in previous work, with the lectins from Solanum tuberosum and Helix pomatia showing particular promise. In this study the acute local dermal irritancy of these lectins, in terms of their potential to cause inflammation and tissue necrosis, was investigated. After an initial study in terminally anaesthetised animals (to ensure no gross toxicity was evident), five male New Zealand white rabbits from the same litter were briefly anaesthetised and Evans blue injected intravenously as a marker of inflammation. Sterile lectin solutions in normal saline at a range of concentrations from 50 to 500 microg ml(-1) were prepared and 50-microl volumes injected intradermally at 18 sites across a shaved area of each rabbit's back. The rabbits were then allowed to regain consciousness. There was no evidence of tissue necrosis, oedema or Evans blue infiltration with any of the lectin solutions administered. The rabbits did not display any signs of discomfort such as scratching or continued grooming throughout the experiment. Histological examination of the injection sites revealed little sign of any inflammation, such as heterophil migration, oedema or tissue damage. It was concluded that these lectins demonstrate minimal acute irritancy, and will, therefore, be taken forward for formulation and in vivo studies.

An atomic force microscopy investigation of bioadhesive polymer adsorption onto human buccal cells.

Atomic force microscopy (AFM) was used to examine the buccal cell surface in order to image the presence of adsorbed bioadhesive polymers identified from previous work. Isotonic saline solution (5 ml) containing either polycarbophil (pH 7.6), chitosan (pH 4.5) or hydroxypropyl methylcellulose (pH 7.6) (0.5% w/v) was exposed to freshly collected buccal cells (ca. 48x10(4) cells/test) for 15 min at 30 degrees C. The cells were then rinsed with a small volume of double distilled water, allowed to air-dry on a freshy cleaved mica surface and imaged using contact mode AFM. Untreated cells showed relatively smooth surface characteristics, with many small 'crater-like' pits and indentations spread over cell surfaces. Cells that had been treated with all the investigated polymers appeared to have lost the crater and indentation characteristic and gained a higher surface roughness. These results suggest that polymer chains had adsorbed onto the cell surfaces. Quantitative image analysis of cell topography showed significant increases (P<0.05) in arithmetic roughness average (R(a)) for all the investigated polymer treated cells surfaces with respect to untreated control specimens. The changes in surface topography indicate the presence of adsorbed polymer, confirming previous work. This study demonstrates the suitability of AFM as a powerful and sensitive technique for detecting and imaging bioadhesive polymers present on mucosal cell surfaces.

An investigation of mucus/polymer rheological synergism using synthesised and characterised poly(acrylic acid)s.

A range of poly(acrylic acid)s with different average degrees of polymerisation and cross-linking densities were synthesised using a solution polymerisation process. The rheological characteristics of aqueous dispersions of these materials and those of mixtures with homogenised pigs gastric mucus were investigated using dynamic oscillatory rheology, and compared to the known mucoadhesive Carbopol 934P. From the storage moduli, the rheological synergy and relative rheological synergy were calculated, and the effects of concentration and pH on this considered. Generally, the larger the molecular weight (and degree of cross-linking), the greater the rheological synergy, with Carbopol 934P giving the most pronounced effect. Rheological synergy was seen to be concentration-dependent, and a maximum concentration to produce an optimum effect was evident. Acid pHs were seen to favour synergy, although in marked contrast to previous literature reports, the optimum mucus-polymer interaction was not observed at the half ionised value (pH = pKa) but at pH regimes that were unique to each polymer type. This could be influenced by the structural constrains imposed on potential hydrogen bonded interactions. It was concluded that synthesising poly(acrylic acid)s with better defined physicochemical properties than commercially available polymers will advance the study of the phenomenon of rheological synergy.

Factors influencing gel-strengthening at the mucoadhesive-mucus interface.

Mechanical spectroscopy was used to examine some of the factors that may affect mucus gel strengthening: the effect of adding various concentrations of sodium chloride; mucoadhesive polymer molecular weight and its concentration; and the introduction of anionic, cationic and neutral polymers. A reduction in the storage modulus of the mucus/mucoadhesive mixture was observed with the introduction of sodium chloride. A poly(acrylic acid) with a molecular weight of 750 kDa gave the optimum mucus gel strengthening effect relative to other molecular weights. An anionic polymer was found to strengthen the mucus gel much more than a neutral or cationic polymer. It was proposed that the gel strengthening effect could be due to the formation of hydrogen bonded intermolecular complexes between the mucoadhesive and the mucus molecules. Furthermore, the complex formed is influenced by the ionic strength of the environment, and the molecular weight, nature and concentration of the mucoadhesive. In all cases the changes in the rheological properties of the mixes could be correlated directly to the strength of mucoadhesion reported in previous studies.

An in-vitro comparison of controlled release aminophylline tablets: Phyllocontin Continus and Pecram.

It has been suggested that two controlled release preparations containing aminophylline, Phyllocontin Continus and Pecram, are clinically equivalent and are therefore interchangeable. In this study, an in-vitro evaluation of the two preparations was completed using the British Pharmacopoeia dissolution apparatus, initially using water and then an acid/buffer medium to provide a similar pH environment to that within the gastrointestinal tract. Similar release profiles were found when water was used as the dissolution medium, with very little variation between tablets within each group. Good fits were obtained for dissolution-controlled release and diffusion-controlled release models. When the acid/buffer solution was used as the dissolution medium a reduction in the rate of release was observed with Phyllocontin. It was predicted that if this was repeated in-vivo then differences in the peak plasma levels between the two formulations would be seen, although these may be masked by the other variables encountered.

An in-vitro investigation of mucosa-adhesive materials for use in controlled drug delivery.

An in-vitro test system was developed to investigate the adhesiveness of various materials to mucus. The results obtained showed good agreement with the findings of previous in-vivo evaluations of mucosa-adhesives. Further investigations found that these materials become adhesive on hydration. Chain length, and the presence of ionizable groups in the molecule, were found to be determinate factors. The physical nature of the gel, and the location at which the mucoadhesive materials hydrated, were of less importance.

Binding of some Diocleinae lectins to the mucosal surfaces of the eye and mouth.

Controlled delivery of diagnostic or therapeutically active agents to the eye and oral cavity is limited by the efficient removal mechanisms that exist in these areas. In this work, recently isolated, purified and characterised Brazilian bean lectins were examined for their ability to bind to the buccal and sublingual mucosa, cornea and conjunctiva of rat, with regard to their potential therapeutic use. These lectins were labelled using biotin (long arm) N-hydroxysuccinimide ester and a technique developed at the University of Portsmouth. Evaluation of binding after 15 min contact with intact (unfixed) rat eye and oral cavity epithelia was completed using a standard histological procedure involving the avidin-biotin reaction and formation of a brown precipitate with a 3,3'-diaminobenzidine solution. When examined by affinity chromatography, all lectins studied were found to show high percentage biotinylation (i.e. less than 4% by weight of total lectin was eluted from the column). The lectin:biotin ratio was estimated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and ranged between 1:2 (Cratylia floribunda) and 1:10.4 (Canavalia brasilensis). As with previous studies, all lectins bound to the mucosal surfaces to some extent--the Cratylia floribunda lectin appearing to be the most promising, whereas the Dioclea violacea lectin showed comparatively weak binding. In all cases, the presence of glucose (the hapten sugar) reduced or eliminated binding. It was concluded that glucose-mannose binding sites are present on these surfaces, and the lectins from C. floribunda showed a particular affinity for these.

Landscaped campus site humanizes life for the elderly.

Conceptual planning and architectural design have enabled the elderly residents at Maple Knoll Village, Springdale, OH, to live somewhat independent lives and to receive essential medical and nursing care. The landscaped campus and linked residential and health care facilities generate a community spirit, an opportunity for social interaction and individual pursuits, and a personal sense of security and physical and emotional well-being.