Non-aqueous silicone elastomer gels as a vaginal microbicide delivery system for the HIV-1 entry inhibitor maraviroc

Aqueous semi-solid polymeric gels, such as those based on hydroxyethylcellulose (HEC) and polyacrylic acid (e.g. Carbopol®), have a long history of use in vaginal drug delivery. However, despite their ubiquity, they often provide sub-optimal clinical performance, due to poor mucosal retention and limited solubility for poorly water-soluble actives. These issues are particularly pertinent for vaginal HIV microbicides, since many lead candidates are poorly water-soluble and where a major goal is the development of a coitally independent, once daily gel product. In this study, we report the use of a non-aqueous silicone elastomer gel for vaginal delivery of the HIV-1 entry inhibitor maraviroc. In vitro rheological, syringeability and retention studies demonstrated enhanced performance for silicone gels compared with a conventional aqueous HEC gel, while testing of the gels in the slug model confirmed a lack of mucosal irritancy. Pharmacokinetic studies following single dose vaginal administration of a maraviroc silicone gel in rhesus macaques showed higher and sustained MVC levels in vaginal fluid, vaginal tissue and plasma compared with a HEC gel containing the same maraviroc loading. The results demonstrate that non-aqueous silicone gels have potential as a formulation platform for coitally independent vaginal HIV microbicides.

Robust spatially resolved pressure measurements using MRI with novel buoyant advection-free preparations of stable microbubbles in polysaccharide gels

MRI of fluids containing lipid coated microbubbles has been shown to be an effective toot for measuring the local fluid pressure. However, the intrinsically buoyant nature of these microbubbles precludes lengthy measurements due to their vertical migration under gravity and pressure-induced coalescence. A novel preparation is presented which is shown to minimize both these effects for at least 25 min. By using a 2% polysaccharide gel base with a small concentration of glycerol and 1,2-distearoyl-sn-glycero-3-phosphocholine coated gas microbubbles, MR measurements are made for pressures between 0.95 and 1.44 bar. The signal drifts due to migration and amalgamation are shown to be minimized for such an experiment whilst yielding very high NMR sensitivities up to 38% signal change per bar.

Antagonistic triblock polymer gels powered by pH oscillations

A study was conducted to create a pH-responsive layer, in which a small change in the individual polyacid or polybase gel length was transferred into a larger motion that curls up the gel. It was observed that the transfer of motion from a linear displacement into a curved displacement through the geometric design effectively increases the displacement rate. A robust, reversible, and chemically driven mechanical actuator was was produced that demonstrated its response over many pH oscillations. The affine nature of the triblock copolymers, demonstrated for for the polyacid and polybase indicated that the effect will also function at some smaller length scales, which is appropriate for a working biomimetic and soft nanotechnology device. The study also demonstrated the potential applicability of these polymeric gels and suggested the fabrication of related molecular machines and devices based on the principles of soft nanotechnology.

Mechanical actuation by responsive polyelectrolyte brushes and triblock gels

Progress in the development of actuating molecular devices based on responsive polymers is reviewed. The synthesis and characterization of "grafted from brushes and triblock copolymers is reported. The responsive nature of polyelectrolyte brushes, grown by surface initiated atomic transfer radical polymerization (ATRP), has been characterized by scanning force microscopy, neutron reflectometry, and single molecule force measurements. The molecular response is measured directly for the brushes in terms of both the brush height and composition and the force generated by a single molecule. Triblock copolymers, based on hydrophobic end blocks and polyacid midblock, have been used to produce polymer gels where the deformation of the molecules can be followed directly by small angle Xray scattering (SAXS), and a correlation between molecular shape change and macroscopic deformation has been established. A Landolt pHoscillator, based on bromate/sulfite/ferrocyanide, with a room temperature period of 20 min and a range of 3.1

Responsive brushes and gels as components of soft nanotechnology

Progress in the development of generic molecular devices based on responsive polymers is discussed. Characterisation of specially synthesised polyelectrolyte gels, "grafted from" brushes and triblock copolymers is reported. A Landolt pH-oscillator, based on bromate/ sulfite/ferrocyanide, with a room temperature period of 20 min and a range of 3.1 gels where the deformation of the molecules can be followed directly by SAXS and a correlation between molecular shape change and macroscopic deformation has been established. The three systems studied allow both the macroscopic and a molecular response to be investigated independently for the crosslinked gels and the brushes. The triblock copolymers demonstrate that the individual response of the polyelectrolyte molecules scale-up to give the macroscopic response of the system in an oscillating chemical reaction.

Grewia gum 2:mucoadhesive properties of compacts and gels

Purpose: To compare the mucoadhesive performance of grewia polysaccharide gum with those of guar gum, carboxymethylcellulose, hydroxypropyl methylcellulose and carbopol 971P. Methods: Grewia polysaccharide gum compacts or gels as well as those of guar gum, carboxymethylcellulose, hydroxypropyl methylcellulose or carbopol 971P were prepared. Texturometric and tensile analysis of the polymer gels and compacts were carried out using a software-controlled penetrometre, TA.XTPlus texture analyzer. The polymer gels were evaluated for hardness, stickiness, work of cohesion and work of adhesion. Furthermore, the detachment force of the polymer compacts from a mucin substrate was evaluated. Results: The work of adhesion of guar gels was significantly greater than that of grewia gels (p < 0.001) but the latter showed a significantly greater work of adhesion than carboxymethylcellulose gels (p < 0.05) and hydroxypropyl methylcellulose gels (p < 0.001). However, the work of cohesion for grewia/mucin gel mixture was significantly greater (p < 0.001) than those of carboxymethylcellulose/mucin, hydroxypropyl methylcellulose/mucin and carbopol 971P/mucin gel blends. The difference between the mucoadhesive performance of grewia compacts and those of hydroxypropyl methylcellulose and carbopol 971P compacts was insignificant (p > 0.05). Conclusion: Grewia polysaccharide gum demonstrated good mucoadhesive properties, comparable to those of carbopol 971P, carboxymethylcellulose, guar gum and hydroxypropyl methylcellulose, and therefore, should be suitable for the formulation of retentive drug delivery devices. © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City.

Strategies and therapeutic opportunities for the delivery of drugs to the esophagus

Targeting of drugs and therapies locally to the esophagus is an important objective in the development of new and more effective dosage forms. Therapies that are retained within the oral cavity for both local and systemic action have been utilized for many years, although delivery to the esophagus has been far less reported. Esophageal disease states, including infections, motility disorders, gastric reflux, and cancers, would all benefit from localized drug delivery. Therefore, research in this area provides significant opportunities. The key limitation to effective drug delivery within the esophagus is sufficient retention at this site coupled with activity profiles to correspond with these retention times; therefore, a suitable formulation needs to provide the drug in a ready-to-work form at the site of action during the rapid transit through this organ. A successfully designed esophageal-targeted system can overcome these obstacles. This review presents a range of dosage form approaches for targeting the esophagus, including bioadhesive liquids and orally retained lozenges, chewing gums, gels, and films, as well as endoscopically delivered therapeutics. The techniques used to measure efficacy both in vitro and in vivo are also discussed. Drug delivery is a growing driver within the pharmaceutical industry and offers benefits both in terms of clinical efficacy, as well as in market positioning, as a means of extending a drug's exclusivity and profitability. Emerging systems that can be used to target the esophagus are reported within this review, as well as the potential of alternative formulations that offer benefits in this exciting area.

Characterisation of protein stability in rod-insert vaginal rings

A major goal in vaccine development is elimination of the 'cold chain', the transport and storage system for maintenance and distribution of the vaccine product. This is particularly pertinent to liquid formulation of vaccines. We have previously described the rod-insert vaginal ring (RiR) device, comprising an elastomeric body into which are inserted lyophilised, rod-shaped, solid drug dosage forms, and having potential for sustained mucosal delivery of biomacromolecules, such as HIV envelope protein-based vaccine candidates. Given the solid, lyophilised nature of these insert dosage forms, we hypothesised that antigen stability may be significantly increased compared with more conventional solubilised vaginal gel format. In this study, we prepared and tested vaginal ring devices fitted with lyophilised rod inserts containing the model antigen bovine serum albumin (BSA). Both the RiRs and the gels that were freeze-dried to prepare the inserts were evaluated for BSA stability using PAGE, turbidimetry, microbial load, MALDI-TOF and qualitative precipitate solubility measurements. When stored at 4°C, but not when stored at 40°C/75% RH, the RiR formulation offered protection against structural and conformational changes to BSA. The insert also retained matrix integrity and release characteristics. The results demonstrate that lypophilised gels can provide relative protection against degradation at lower temperatures compared to semi-solid gels. The major mechanism of degradation at 40°C/75% RH was shown to be protein aggregation. Finally, in a preliminary study, we found that addition of trehalose to the formulation significantly reduces the rate of BSA degradation compared to the original formulation when stored at 40°C/75% RH. Establishing the mechanism of degradation, and finding that degradation is decelerated in the presence of trehalose, will help inform further development of RiRs specifically and polymer based freeze-dried systems in general.

Optical fiber cavity ring down measurement of refractive index with a microchannel drilled by femtosecond laser

A microchannel was inscribed in the fiber of a ring cavity which was constructed using two 0.1%:99.9% couplers and a 10-m fiber loop. Cavity ring down spectroscopy was used to measure the refractive index (RI) of gels infused into the microchannel. The ring down time discloses a nonlinear increase with respect to RI of the gel and sensitivity up to 300 µs/RI unit and an index resolution of 1.4 × 10 was obtained. © 2009 IEEE.

Finitely coordinated models for low-temperature phases of amorphous systems

We introduce models of heterogeneous systems with finite connectivity defined on random graphs to capture finite-coordination effects on the low-temperature behaviour of finite-dimensional systems. Our models use a description in terms of small deviations of particle coordinates from a set of reference positions, particularly appropriate for the description of low-temperature phenomena. A Born-von Karman-type expansion with random coefficients is used to model effects of frozen heterogeneities. The key quantity appearing in the theoretical description is a full distribution of effective single-site potentials which needs to be determined self-consistently. If microscopic interactions are harmonic, the effective single-site potentials turn out to be harmonic as well, and the distribution of these single-site potentials is equivalent to a distribution of localization lengths used earlier in the description of chemical gels. For structural glasses characterized by frustration and anharmonicities in the microscopic interactions, the distribution of single-site potentials involves anharmonicities of all orders, and both single-well and double-well potentials are observed, the latter with a broad spectrum of barrier heights. The appearance of glassy phases at low temperatures is marked by the appearance of asymmetries in the distribution of single-site potentials, as previously observed for fully connected systems. Double-well potentials with a broad spectrum of barrier heights and asymmetries would give rise to the well-known universal glassy low-temperature anomalies when quantum effects are taken into account. © 2007 IOP Publishing Ltd.

Enzymatic stabilization of gelatin-based scaffolds

The definitive goal of this research is to develop protein-based scaffolds for use in soft tissue regeneration, particularly in the field of dermal healing. The premise of this investigation was to characterize the mechanical properties of gelatin cross-linked with microbial transglutaminase (mTGase) and to investigate the cytocompatibility of mTGase cross-linked gelatin. Dynamic rheological analysis revealed a significant increase in the storage modulus and thermal stability of gelatin after cross-linking with mTGase. Static, unconfined compression tests showed an increase in Young's modulus of gelatin gels after mTGase cross-linking. A comparable increase in gel strength was observed with 0.03% mTGase and 0.25% glutaraldehyde cross-linked gelatin gels. In vitro studies using 3T3 fibroblasts indicated cytotoxicity at a concentration of 0.05% mTGase after 72 h. However, no significant inhibition of cell proliferation was seen with cells grown on lower concentrations of mTGase cross-linked gelatin substrates. The mechanical improvement and cytocompatibility of mTGase cross-linked gelatin suggests mTGase has potential for use in stabilizing gelatin gels for tissue-engineering applications.

Purification of plasmid DNA for use in human gene therapy

Two key issues defined the focus of this research in manufacturing plasmid DNA for use In human gene therapy. First, the processing of E.coli bacterial cells to effect the separation of therapeutic plasmid DNA from cellular debris and adventitious material. Second, the affinity purification of the plasmid DNA in a Simple one-stage process. The need arises when considering the concerns that have been recently voiced by the FDA concerning the scalability and reproducibility of the current manufacturing processes in meeting the quality criteria of purity, potency, efficacy, and safety for a recombinant drug substance for use in humans. To develop a preliminary purification procedure, an EFD cross-flow micro-filtration module was assessed for its ability to effect the 20-fold concentration, 6-time diafiltration, and final clarification of the plasmid DNA from the subsequent cell lysate that is derived from a 1 liter E.coli bacterial cell culture. Historically, the employment of cross-flow filtration modules within procedures for harvesting cells from bacterial cultures have failed to reach the required standards dictated by existing continuous centrifuge technologies, frequently resulting in the rapid blinding of the membrane with bacterial cells that substantially reduces the permeate flux. By challenging the EFD module, containing six helical wound tubular membranes promoting centrifugal instabilities known as Dean vortices, with distilled water between the Dean number's of 187Dn and 818Dn,and the transmembrane pressures (TMP) of 0 to 5 psi. The data demonstrated that the fluid dynamics significantly influenced the permeation rate, displaying a maximum at 227Dn (312 Imh) and minimum at 818Dn (130 Imh) for a transmembrane pressure of 1 psi. Numerical studies indicated that the initial increase and subsequent decrease resulted from a competition between the centrifugal and viscous forces that create the Dean vortices. At Dean numbers between 187Dn and 227Dn , the forces combine constructively to increase the apparent strength and influence of the Dean vortices. However, as the Dean number in increases above 227 On the centrifugal force dominates the viscous forces, compressing the Dean vortices into the membrane walls and reducing their influence on the radial transmembrane pressure i.e. the permeate flux reduced. When investigating the action of the Dean vortices in controlling tile fouling rate of E.coli bacterial cells, it was demonstrated that the optimum cross-flow rate at which to effect the concentration of a bacterial cell culture was 579Dn and 3 psi TMP, processing in excess of 400 Imh for 20 minutes (i.e., concentrating a 1L culture to 50 ml in 10 minutes at an average of 450 Imh). The data demonstrated that there was a conflict between the Dean number at which the shear rate could control the cell fouling, and the Dean number at which tile optimum flux enhancement was found. Hence, the internal geometry of the EFD module was shown to sub-optimal for this application. At 579Dn and 3 psi TMP, the 6-fold diafiltration was shown to occupy 3.6 minutes of process time, processing at an average flux of 400 Imh. Again, at 579Dn and 3 psi TMP the clarification of the plasmid from tile resulting freeze-thaw cell lysate was achieved at 120 Iml1, passing 83% (2,5 mg) of the plasmid DNA (6,3 ng μ-1 10.8 mg of genomic DNA (∼23,00 Obp, 36 ng μ-1 ), and 7.2 mg of cellular proteins (5-100 kDa, 21.4 ngμ-1 ) into the post-EFD process stream. Hence the EFD module was shown to be effective, achieving the desired objectives in approximately 25 minutes. On the basis of its ability to intercalate into low molecular weight dsDNA present in dilute cell lysates, and be electrophoresed through agarose, the fluorophore PicoGreen was selected for the development of a suitable dsDNA assay. It was assesseel for its accuracy, and reliability, In determining the concentration and identity of DNA present in samples that were eleclrophoresed through agarose gels. The signal emitted by intercalated PicoGreen was shown to be constant and linear, and that the mobility of the PicaGreen-DNA complex was not affected by the intercalation. Concerning the secondary purification procedure, various anion-exchange membranes were assessed for their ability to capture plasmid DNA from the post-EFD process stream. For a commercially available Sartorius Sartobind Q15 membrane, the reduction in the equilibriumbinding capacity for  ctDNA in buffer of increasing ionic demonstrated that DNA was being.adsorbed by electrostatic  interactions only. However, the problems associated with fluid distribution across the membrane demonstrated that the membrane housing was the predominant cause of the .erratic breakthrough curves. Consequently, this would need to be rectified before such a membrane could be integrated into the current system, or indeed be scaled beyond laboratory scale. However, when challenged with the process material, the data showed that considerable quantities of protein (1150 μg) were adsorbed preferentially to the plasmid DNA (44 μg). This was also shown for derived Pall Gelman UltraBind US450 membranes that had been functionalised by varying molecular weight poly-L~lysine and polyethyleneimine ligands. Hence the anion-exchange membranes were shown to be ineffective in capturing plasmid DNA from the process stream. Finally, work was performed to integrate a sequence-specific DNA·binding protein into a single-stage DNA chromatography, isolating plasmid DNA from E.coli cells whilst minimising the contamination from genomic DNA and cellular protein. Preliminary work demonstrated that the fusion protein was capable of isolating pUC19 DNA into which the recognition sequence for the fusion-protein had been inserted (pTS DNA) when in the presence of the conditioned process material. Althougth the pTS recognition sequence differs from native pUC19 sequences by only 2 bp, the fusion protein was shown to act as a highly selective affinity ligand for pTS DNA alone. Subsequently, the scale of the process was scaled 25-fold and positioned directly following the EFD system. In conclusion, the integration of the EFD micro-filtration system and zinc-finger affinity purification technique resulted in the capture of approximately 1 mg of plasmid DNA was purified from 1L of E.coli  culture in a simple two stage process, resulting in the complete removal of genomic DNA and 96.7% of cellular protein in less than 1 hour of process time.

Novel cationic polymers for use at biological interfaces

One of the main problems with the use of synthetic polymers as biomaterials is the invasion of micro-organisms causing infection. A study of the properties of polymeric antibacterial agents, in particular polyhexamethylene biguanide, has revealed that the essential components for the design of a novel polymeric antibacterial are a balance between hydrophilicity and hydrophobicity coupled with sites of cationicity. The effect of cation incorporation on the physical properties of hydrogels has been investigated. Hydrogel systems copolymerised with either N-vinyl imidazole or dimethylaminoethyl methacrylate have been characterised in terms of their water binding, mechanical and surface properties. It has been concluded that the incorporation of these monomers does not adversely affect the properties of such hydrogels and that these materials are potential candidates for further development for use in biomedical applications. It has been reported that hydro gels with ionic character may increase the deposition of biological material onto the hydrogel surface when it is in contact with body fluids. An investigation into the deposition characteristics of hydrogels containing the potentially cationic monomers has been carried out, using specific protein adsorption and in vitro spoilation techniques. The results suggest that at low levels of cationicity, the deposition of positively charged proteins is reduced without adversely affecting the uptake of the other proteins. The gross deposition characteristics were found to be comparable to some commercially available contact lens materials. A preliminary investigation into the development of novel antibacterial polymers has been completed and some novel methods of bacterial inhibition discussed. These methods include development of an hydrogel whose potential application is as a catheter coating.

Novel hydrogel polymers

Hydrogels may be conveniently described as hydrophilic polymers that are swollen by, but do not dissolve in water. In this work a series of copolymer hydrogels and semi-interpenetrating polymer networks based on the monomers 2-hydroxyethyl methacrylate, N-vinyl pyrrolidone and N'N' dimethyl acrylamide, together with some less hydrophilic hydroxyalkyl acrylates and methacrylates have been synthesised. Variations in structure and composition have been correlated both with the total equilibrium water content of the resultant hydrogel and with the more detailed water binding behaviour, as revealed by differential scanning calorimetry studies. The water binding characteristics of the hydrogels were found to be primarily a function of the water structuring groups present in gel. The water binding abilities of these groups were, however, modified by steric effects. The mechanical properties of the hydrogels were also investigated. These were found to be dependent on both the polymer composition and the amount and nature of the water present in the gels. In biological systems, composite formation provides a means of producing strong, high water content materials. As an analogy with these systems hydrogel composites were prepared. In an initial study of these materials the water binding and mechanical properties of semi-interpenetrating polymer networks of N'N'dimethyl acrylamide with cellulosic type materials, with polyurethanes and with ester containing polymers were examined. A preliminary investigation of surface properties of both the copolymers and semi-interpenetrating polymer networks has been completed, using both contact angle measurements and anchorage dependent fibroblast cells. Measurable differences in surface properties attributable to structural variations in the polymers were detected by droplet techniques in the dehydrated state. However, in the hydrated state these differences were masked by the water in the gels. The use of cells enabled the underlying differences to be probed and the nature of the water structuring group was again found to be the dominant factor.

Design and synthesis of novel hydrogels for biological applications

The aims of this project were:1) the synthesis of a range of new polyether-based vinylic monomers and their incorporation into poly(2-hydroxyethyl methacrylate) (poly(HEMA)) based hydrogel networks, of interest to the contact lens industry.2) the synthesis of a range of alkyltartronic acids, and their derivatives. These molecules may ultimately be used to produce functionalised poly(-hydroxy acids) of potential interest in either drug delivery or surgical suture applications. The novel syntheses of a range of both methoxy poly(ethylene glycol) acrylates (MPEGAs) and poly(ethylene glycol) acrylates (PEGAs) are described. Products were obtained in very good yields. These new polyether-based vinylic monomers were copolymerised with 2-hydroxyethyl methacrylate (HEMA) to produce a range of hydrogels. The equilibrium water contents (EWC) and surface properties of these copolymers containing linear polyethers were examined. It was found that the EWC was enhanced by the presence of the hydrophilic polyether chains.Results suggest that the polyether side chains express themselves at the polymer surface, thus dictating the surface properties of the gels. Consequentially, this leads to an advantageous reduction in the surface adhesion of biological species. A synthesis of a range of alkyltartronic acids is also described. The acids prepared were obtained in very good yields using a novel four-stage synthesis. These acids were modified to give potassium monoethyl alkyltartronates. Although no polyesterification is described in this thesis, these modified alkyltartronic acid derivatives are considered to be potentially excellent starting materials for poly (alkyltartronic acid) synthesis via anhydrocarboxylate or anhydrosulphite cyclic monomers.