Effect of serum concentration on the cytotoxicity of clay particles.

Nanoparticle cytotoxicity testing based on in vitro methods frequently lack consistency. Even the inclusion of the commonly employed growth supplement, FCS (fetal calf serum), generates variable results. Thus, our object was to investigate the effect of FCS concentration on the cytotoxic behaviour of the unmodified nanoclay, Cloisite® Na(+). Human monocytic U937 cells in medium supplemented with 5% FCS, 2.5% FCS or serum-free medium were treated with 1 mg/ml Cloisite Na(+). Cell growth in 2.5% FCS was significantly inhibited by Cloisite Na(+) within 48 h, whereas little effect was seen with a supplement of 5% FCS. Without serum, cell growth was inhibited and Cloisite Na(+) had a detrimental effect on these cells. In media supplemented with FCS, the nanoclays agglomerated together to form large bundles, whereas they were evenly dispersed throughout the medium in the absence of serum. Clay particles, therefore, have cytotoxic properties that may be linked to their dispersion pattern. These adverse effects seem to be masked by 5% FCS. Serum supplementation is an important consideration in the toxicological assessments of nanomaterials on cells, which needs to be addressed in the standardization of in vitro testing methods.

Cytotoxic effects induced by unmodified and organically modified nanoclays in the human hepatic HepG2 cell line.

The term 'nanoclay' generically refers to the natural clay mineral, montmorillonite, with silica and alumina as the dominant constituents. The incorporation of nanoclays into polymeric systems dramatically enhances their barrier properties as well as their thermal and mechanical resistance. Consequently, nanoclays are employed in a wide range of industrial applications with recent studies reporting potential use in the modulation of drug release. With the increase in manufacturing of nanoclay-containing products, information on the toxicological and health effects of nanoclay exposure is warranted. Thus, the objective of the present study was to evaluate the cytotoxicity of two different nanoclays: the unmodified nanoclay, Cloisite Na+ ®, and the organically modified nanoclay, Cloisite 93A®, in human hepatoma HepG2 cells. Following 24 h exposure the nanoclays significantly decreased cell viability. Cloisite Na+ induced intracellular reactive oxygen species (ROS) formation which coincided with increased cell membrane damage, whilst ROS generation did not play a role in Cloisite 93A-induced cell death. Neither of the nanoclays induced caspase-3/7 activation. Moreover, in the cell culture medium the nanoclays aggregated differently and this appeared to have an effect on their mechanisms of toxicity. Taken together, our data demonstrate that nanoclays are highly cytotoxic and as a result pose a possible risk to human health.

The Effect of Cooling on the Degree of Crystallinity, Solid-State Properties, and Dissolution Rate of Multi-Component Hot-Melt Extruded Solid Dispersions.

: The effect of cooling on the degree of crystallinity, solid-state and dissolution properties of multi-component hot-melt extruded solid dispersions [SD] is of great interest for the successful formulation of amorphous SDs and is an area that is unreported, especially in the context of improving the stability of these specific systems. The thermal solid-state properties, degree of crystallinity, drug-polymer interactions, solubility and physical stability over time were investigated. X-ray powder diffraction [XRPD] and hyper differential scanning calorimetry [DSC] confirmed that indomethacin [INM] was converted to the amorphous state; however, the addition of poloxamer 407 [P407] had a significant effect on the degree of crystallinity and the solubility of the SD formulations. Spectroscopy studies identified the mechanism of interaction and solubility studies, showing a higher dissolution rate compared to amorphous and pure INM in pH 1.2 with a kinetic solubility of 20.63 µg/mL and 34.7 µg/mL after 3 and 24 h. XRPD confirmed that INM remained amorphous after 5 months stability testing in solid solutions with Poly(vinylpyrrolidone-co-vinyl acetate) [PVP VA64] and Plasdone S-630 [PL-S630]. Although cooling had a significant effect on the degree of crystallinity and on solubility of INM, the cooling method used did not have any significant effect on the amorphous stability of INM over time.

Development of a novel porous cryo-foam for potential wound healing applications.

This body of work describes the development of a porous hydrogel for wound healing applications. In the present study poly (vinyl alcohol) (PVA) and poly (acrylic acid) (PAA) based hydrogels were prepared, and their properties were examined. Varying concentrations of the polymers and distilled water were used to prepare the hydrogels. The use of a high shear mixer, for foaming the PVA and PVA/PAA gels, and how this physical change can affect the structure and porosity of the hydrogel in question, represents a key feature of this work. The mechanical and thermal properties were determined by parallel plate rheometry and modulated differential scanning calorimetry (MDSC) respectively. The results indicated that the hydrogels containing low concentration of PVA and high volume of H(2)O are significantly weaker than those synthesised with higher concentrations of PVA. The thermal analysis shows distinct endotherms and provides evidence of crystallisation. The chemical structure of the hydrogels was confirmed by means of attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR).

Evaluation of the materials properties, stability and cell response of a range of PEGDMA hydrogels for tissue engineering applications.

The main aim of this study was to examine the stability of a range of polyethyleneglycol dimethacrylate (PEGDMA) hydrogels over a 28-day period in simulated physiological solution. Upon optimisation of the ultraviolet (UV) curing conditions, the PEGDMA hydrogels were prepared using four different monomer concentrations (25, 50, 75 and 100 wt% PEGDMA) in water and cross-linked by photopolymerisation. Initial results revealed a correlation between monomer concentration and swelling behaviour, where a decrease in swelling was observed with increase in monomer content. On storage in physiological solutions at 37 °C, a decrease in the weight remaining of the hydrogels and the pH of the solutions was observed over a 28-day period. Using scanning electron microscopy, the surface topography of the hydrogels appeared to get smoother and in parallel changes in hydrophilicty were observed, with the biggest changes observed for the higher monomer concentrations where water contact angle values were seen to increase toward 90°. However, the mechanical properties remained relatively unaffected and there was no adverse effect on cell metabolic activity observed for cells grown in the presence of PEGDMA samples or using elution methods. Looking at the combination of mechanical chemical and thermal properties shown these results are an important finding for scaffolds intended for tissue engineering applications, where provision of mechanical support without the elicitation of an inflammatory response due to polymer degradation products is crucial for successful integration and neotissue formation during the first 28 days post implantation.

Additive Manufacturing of Personalized Pharmaceutical Dosage Forms via Stereolithography.

The introduction of three-dimensional printing (3DP) has created exciting possibilities for the fabrication of dosage forms, paving the way for personalized medicine. In this study, oral dosage forms of two drug concentrations, namely 2.50% and 5.00%, were fabricated via stereolithography (SLA) using a novel photopolymerizable resin formulation based on a monomer mixture that, to date, has not been reported in the literature, with paracetamol and aspirin selected as model drugs. In order to produce the dosage forms, the ratio of poly(ethylene glycol) diacrylate (PEGDA) to poly(caprolactone) triol was varied with diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (Irgacure TPO) utilized as the photoinitiator. The fabrication of 28 dosages in one print process was possible and the printed dosage forms were characterized for their drug release properties. It was established that both drugs displayed a sustained release over a 24-h period. The physical properties were also investigated, illustrating that SLA affords accurate printing of dosages with some statistically significant differences observed from the targeted dimensional range, indicating an area for future process improvement. The work presented in this paper demonstrates that SLA has the ability to produce small, individualized batches which may be tailored to meet patients' specific needs or provide for the localized production of pharmaceutical dosage forms.

Micro-Injection Moulding of Poly(vinylpyrrolidone-vinyl acetate) Binary and Ternary Amorphous Solid Dispersions.

Micro-injection moulding (µIM) was used for the production of enteric tablets of plasticised and unplasticised solid dispersions of poly(vinylpyrrolidone-vinyl acetate) (PVPVA), and the effect of the mechanical and thermal treatment on the properties of the dispersions was investigated. The physical state of the systems showed to be unaltered by the µIM step, maintaining the drug in the amorphous state. The dissolution profile of the tablets showed a slower dissolution rate due to the lower surface to volume ratio compared to the extruded strands. The lack of solubility of the doses in the acidic medium as a consequence of the acidity of indomethacin (IND) was observed. However, in neutral pH the drug dissolution showed slower rates without affecting the dissolution extent, showing a potential application for the development of controlled release doses. Overall, the production of tablets of amorphous solid dispersions (ASD), coupling hot-melt extrusion (HME) and µIM, proved to be a successful approach towards a continuous automated manufacturing process to improve the aqueous solubility of poorly water-soluble drugs.

Characterisation and controlled drug release from novel drug-loaded hydrogels.

Hydrogel based devices belong to the group of swelling controlled drug delivery systems. Temperature responsive poly(N-isopropylacrylamide)-poly(vinylpyrrolidinone) random copolymers were produced by free radical polymerisation, using 1-hydroxycyclohexylphenyketone as an ultraviolet-light sensitive initiator, and poly(ethylene glycol) dimethacrylate as the crosslinking agent (where appropriate). The hydrogels were synthesised to have lower critical solution temperatures (LCST) near body temperature, which is favourable particularly for 'smart' drug delivery applications. Two model drugs (diclofenac sodium and procaine HCl) were entrapped within these xerogels, by incorporating the active agents prior to photopolymerisation. The properties of the placebo samples were contrasted with the drug-loaded copolymers at low levels of drug integration. Modulated differential scanning calorimetry (MDSC), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and atomic force microscopy (AFM) were used to investigate the influence of the drugs incorporated on the solid-state properties of the xerogels. MDSC and swelling studies were carried out to ascertain their effects on the LCST and swelling behaviour of the hydrated samples. In all cases, drug dissolution analysis showed that the active agent was released at a slower rate at temperatures above the phase transition temperature. Finally, preliminary in vitro cytotoxicity evaluations were performed to establish the toxicological pattern of the gels.

The significance of variation in extrusion speeds and temperatures on a PEO/PCL blend based matrix for oral drug delivery.

The body of work described in this research paper outlines the use of PEO/PCL blends in the production of monolithic matrices for oral drug delivery. Several batches of matrix material were prepared with carvedilol used as the active pharmaceutical ingredient. The matrices were prepared using various extrusion parameters to investigate the effect of screw speed and barrel temperature on the properties of the drug delivery devices. The resultant extrudate was characterised using steady state parallel plate rheometry, differential scanning calorimetry (DSC) and dissolution testing. Higher screw speeds were observed to result in slightly lower matrix melt viscosity when compared with matrices compounded using lower screw speeds. Dissolution testing showed that the incorporation of the hydrophobic PCL polymer into a PEO matrix results in a retarded drug release profile.

Investigation of miscibility estimation methods between indomethacin and poly(vinylpyrrolidone-co-vinyl acetate).

The investigation of the miscibility between active pharmaceutical ingredients (API's) and polymeric excipients is of great interest for the formulation and development of amorphous solid dispersions, especially in the context of the prediction of the stability of these systems. Two different methods were applied to determine the miscibility between model compounds poly(vinylpyrrolidone-co-vinyl acetate) (PVPVA) and indomethacin (IND), viz. the measurement of the glass transition temperature (Tg) and the melting point depression method framed on the Flory-Huggins theory. Measurement of the glass transition temperatures of the binary blends showed the formation of an amorphous single phase system between the PVPVA and the IND regardless of the composition. Variation of Tg with the composition was well described by the Gordon-Taylor equation leading to the error of concluding lack of intermolecular interactions between the materials. Application of the Brostow-Chiu-Kalogeras-Vassilikou-Dova (BCKV) model shows a negative interaction parameter (a0) suggesting the presence of drug-drug intermolecular interactions. Application of the melting point depression method within the framework of the Flory-Huggins theory proved the miscibility of the system at temperatures close to the melting point of IND.

Investigation of Ethylene Oxide-co-propylene Oxide for Dissolution Enhancement of Hot-Melt Extruded Solid Dispersions.

The optimal design of amorphous solid dispersion formulations requires the use of excipients to maintain supersaturation and improve physical stability to ensure shelf-life stability and better absorption during intestinal transit, respectively. Blends of excipients (surfactants and polymers) are often used within pharmaceutical products to improve the oral delivery of Biopharmaceutical Classification System class II drugs. Therefore, in this study, a dissolution enhancer, poloxamer 407 (P407), was investigated to determine its effect on the dissolution properties and on the amorphous nature of the active pharmaceutical ingredient contained in the formulation. Phase solubility studies of indomethacin (INM) in aqueous solutions of P407 and poly(vinylpyrrolidone-vinyl acetate copolymer) showed an increase in the kinetic solubility of INM compared with the pure drug at 37°C with a Ka value of 0.041 µg/mL. The solid dispersions showed a higher dissolution rate when compared to pure and amorphous drugs when performed in pH buffer 1.2 with a kinetic solubility of 21 µg/mL. The stability data showed that the amorphous drug in solid solutions with poly(vinylpyrrolidone-vinyl acetate copolymer) and P407 remained amorphous, and the %P407 loading had no effect on the amorphous stability of INM. This study concluded that the amorphous solid dispersion contributed to the increased solubility of INM.

Preparation of a novel freeze thawed poly(vinyl alcohol) composite hydrogel for drug delivery applications.

We describe a drug delivery system based on a physically cross-linked poly(vinyl alcohol) (PVA) hydrogel for the release of Theophylline (TH). A composite was created by freezing an aqueous solution of PVA/NaOH onto a PVA/poly(acrylic acid) substrate. This formed a strong interface and demonstrated greater physical strength than the hydrogel alone. Such systems have potential for a variety of localised controlled drug delivery applications, for example, as coatings for implantable devices. Importantly, the results suggest that a versatile synthetic platform is possible that may provide different functional materials or combination of such. The resultant samples were characterised using optical microscopy, modulated differential scanning calorimetry (MDSC) and dissolution testing. The microstructure of the gels was examined using micro-thermal analysis (microTA) which is a combination of atomic force microscopy and thermal analysis. TH was found to have an effect on the crystalline structure and dissolution showed a Fickian release, suggesting that swelling and crystallinity were the controlling mechanisms.

Preparation of monolithic matrices for oral drug delivery using a supercritical fluid assisted hot melt extrusion process.

The use of supercritical fluids as plasticisers in polymer processing has been well documented. The body of work described in this research paper outlines the use of a supercritical CO(2) assisted extrusion process in the preparation of a hot melt extruded monolithic polymer matrix for oral drug delivery. Several batches of matrix material were prepared with Carvedilol used as the active pharmaceutical ingredient (API). These batches were subsequently extruded both with and without supercritical CO(2) incorporation. The resultant matrices were characterised using steady-state parallel plate rheometry, differential scanning calorimetry (DSC), atomic force microscopy (AFM), micro-thermal analysis (microTA) and dissolution testing. Dissolution analysis showed that the use of supercritical CO(2) during the extrusion process resulted in a faster dissolution of API when compared with unassisted extrusion. The supercritical CO(2) incorporation also resulted in reduced viscosity during processing, therefore allowing for quicker throughput and productivity. The results detailed within this paper indicate that supercritical fluid assisted hot melt extrusion is a viable enhancement to conventional hot melt extrusion for the production of monolithic dosage forms.

The use of agar as a novel filler for monolithic matrices produced using hot melt extrusion.

The use of filler materials in an extended release monolithic polymer matrix can lead to a vastly altered release profile for the active pharmaceutical ingredient. A range of excipients for use in monolithic matrices have been discussed in the literature. The body of work described in this research paper outlines the use of agar as a novel filler material in a hot melt extruded polymer matrix. Several batches of matrix material were prepared with Diclofenac sodium used as the active pharmaceutical ingredient (API). Agar and microcrystalline cellulose were used as the filler materials in varying ratios, to examine the effect of % filler content as well as filler type on the properties of the hot melt extruded matrix. The resultant extrudates were characterised using steady state parallel plate rheometry, differential scanning calorimetry (DSC) and dissolution testing. The rheometry analysis concluded that the fillers used resulted in an increase in the matrix viscosity. The DSC scans obtained showed negligible effects on the melting behavior of the matrix as a result of the filler inclusion. Dissolution analysis showed that the presence of the fillers resulted in a slower release rate of API than for the matrix alone. The results detailed within this paper indicate that agar is a viable filler for extended release hot melt produced dosage forms.

Multifunctional polyvinylpyrrolidinone-polyacrylic acid copolymer hydrogels for biomedical applications.

Copolymers of N-vinylpyrrolidinone and acrylic acid, crosslinked with ethylene glycol dimethacrylate and polyethylene glycol 600 dimethacrylate were prepared by UV-polymerisation. These polymers were analysed for their extractable content by Soxhlet extraction of the samples at 100 degrees C for 72 h. Aspirin and paracetamol were incorporated into the polymer structure at 25 wt.% during the curing process and their presence confirmed by Fourier transform infrared spectroscopy. It was found that the release rate of the drug from the polymer matrix was dependent on intermolecular bonding between the polymer and active agent with aspirin being released slower than paracetamol in all cases. Results showed that paracetamol was completely released after 24h whereas complete release of aspirin took up to 70 h. Finally preliminary in vitro biocompatibility testing was performed for crosslinked polyvinylpyrrolidinone, by determining human hepatoma HepG2 cell viability in the MTT assay and DNA damage in the comet assay following direct contact with various concentrations of polyvinylpyrrolidinone-containing media. Cytotoxicity data suggests a dose-dependent effect for both crosslinkers, with concentrations in the range 0.025-2.5 mg ml(-1) showing a marginal decrease in viability to, at most, 70% that of untreated cells. Again DNA migration in the comet assay following short-term exposure to EGDMA crosslinked hydrogels correlates with MTT data.

Fabrication and in vitro biological evaluation of photopolymerisable hydroxyapatite hydrogel composites for bone regeneration.

The aim of this study was to improve the bioactive and compressive properties of photopolymerisable polyethylene glycol hydrogels with the incorporation of hydroxyapatite at different loadings. The synthesis of pure hydroxyapatite was verified through Fourier transform infrared spectroscopy (FTIR) analysis by the complete reaction of all constituents. The formation of a bioactive layer of the hydrogel based composites was confirmed through the formation of carbonate hydroxyapatite after soaking the samples in simulated body fluid. The incorporation of hydroxyapatite into the system resulted in an increase in Young's modulus from 4.36 to 12.73 MPa and an increase in the stress at limit value from 1.20 to 4.42 MPa. This was due to the hydroxyapatite absorbing the compressive load, the polymer matrix distributing the load, a reduction in swelling and the presence of physical crosslinking between both components. Drug dissolution testing showed that the release rate of a drug from the hydrogels was dependent on the molecular weight of the polymer and the type of drug used.

Effects of temperature, packaging and electron beam irradiation processing conditions on the property behaviour of Poly (ether-block-amide) blends.

The radiation stability of Poly (ether-block-amide) (PEBA) blended with a multifunctional phenolic antioxidant and a hindered amide light stabiliser was examined under various temperatures, packaging and electron beam processing conditions. FTIR revealed that there were slight alterations to the PEBA before irradiation; however, these became more pronounced following irradiation. The effect of varying the temperature, packaging and processing conditions on the resultant PEBA properties was apparent. For example, rheology demonstrated that the structural properties could be enhanced by manipulating the aforementioned criteria. Mechanical testing exhibited less radiation resistance when the PEBA samples were vacuum packed and exposed to irradiation. MFI and AFM confirmed that the melting strength and surface topography could be reduced/increased depending on the conditions employed. From this study it was concluded that virgin PEBA submerged in dry ice with non-vacuum packaging during the irradiation process, provided excellent radiation resistance (20.9% improvement) in contrast to the traditional method.

Effects of gamma ray and electron beam irradiation on the mechanical, thermal, structural and physicochemical properties of poly (ether-block-amide) thermoplastic elastomers.

Both gamma ray and electron beam irradiation are widely used as a means of medical device sterilisation. However, it is known that the radiation produced by both processes can lead to undesirable changes within biomedical polymers. The main objective of this research was to conduct a comparative study on the two key radiosterilisation methods (gamma ray and electron beam) in order to identify the more detrimental process in terms of the mechanical, structural, chemical and thermal properties of a common biomedical grade polymer. Poly (ether-block-amide) (PEBA) was prepared by injection moulding ASTM testing specimens and these were exposed to an extensive range of irradiation doses (5-200 kGy) in an air atmosphere. The effect of varying the irradiation dose concentration on the resultant PEBA properties was apparent. For instance, the tensile strength, percentage elongation at break and shore D hardness can be increased/decreased by controlling the aforementioned criteria. In addition, it was observed that the stiffness of the material increased with incremental irradiation doses as anticipated. Melt flow index demonstrated a dramatic increase in the melting strength of the material indicating a sharp increase in molecular weight. Conversely, modulated differential scanning calorimetry established that there were no significant alterations to the thermal transitions. Noteworthy trends were observed for the dynamic frequency sweeps of the material, where the crosslink density increased according to an increase in electron beam irradiation dose. Trans-vinylene unsaturations and the carbonyl group concentration increased with an increment in irradiation dose for both processes when observed by FTIR. The relationship between the irradiation dose rate, mechanical properties and the subsequent surface properties of PEBA material is further elucidated throughout this paper. This study revealed that the gamma irradiation process produced more adverse effects in the PEBA material in contrast to the electron beam irradiation process.

Hydrogel/bioactive glass composites for bone regeneration applications: synthesis and characterisation.

Due to the deficiencies of current commercially available biological bone grafts, alternative bone graft substitutes have come to the forefront of tissue engineering in recent times. The main challenge for scientists in manufacturing bone graft substitutes is to obtain a scaffold that has sufficient mechanical strength and bioactive properties to promote formation of new tissue. The ability to synthesise hydrogel based composite scaffolds using photopolymerisation has been demonstrated in this study. The prepared hydrogel based composites were characterised using techniques including Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), Energy-dispersive X-ray spectrometry (EDX), rheological studies and compression testing. In addition, gel fraction, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), porosity and swelling studies of the composites were carried out. It was found that these novel hydrogel bioglass composite formulations did not display the inherent brittleness that is typically associated with bioactive glass based bone graft materials and exhibited enhanced biomechanical properties compared to the polyethylene glycol hydrogel scaffolds along. Together, the combination of enhanced mechanical properties and the deposition of apatite on the surface of these hydrogel based composites make them an ideal candidate as bone graft substitutes in cancellous bone defects or low load bearing applications.

Cell encapsulation and cryostorage in PVA-gelatin cryogels: incorporation of carboxylated ε-poly-L-lysine as cryoprotectant.

It is desirable to produce cryopreservable cell-laden tissue-engineering scaffolds whose final properties can be adjusted during the thawing process immediately prior to use. Polyvinyl alcohol (PVA)-based solutions provide platforms in which cryoprotected cell suspensions can be turned into a ready-to-use, cell-laden scaffold by a process of cryogelation. In this study, such a PVA system, with DMSO as the cryoprotectant, was successfully developed. Vascular smooth muscle cell (vSMC)-encapsulated cryogels were investigated under conditions of cyclic strain and in co-culture with vascular endothelial cells to mimic the environment these cells experience in vivo in a vascular tissue-engineering setting. In view of the cytotoxicity DMSO imposes with respect to the production procedure, carboxylated poly-L-lysine (COOH-PLL) was substituted as a non-cytotoxic cryoprotectant to allow longer, slower thawing periods to generate more stable cryogels. Encapsulated vSMC with DMSO as a cryoprotectant responded to 10% cyclic strain with increased alignment and proliferation. Cells were stored frozen for 1 month without loss of viability compared to immediate thawing. SMC-encapsulated cryogels also successfully supported functional endothelial cell co-culture. Substitution of COOH-PLL in place of DMSO resulted in a significant increase in cell viability in encapsulated cryogels for a range of thawing periods. We conclude that incorporation of COOH-PLL during cryogelation preserved cell functionality while retaining fundamental cryogel physical properties, thereby making it a promising platform for tissue-engineering scaffolds, particularly for vascular tissue engineering, or cell preservation within microgels.