Biocomposites of non-crosslinked natural and synthetic polymers

Biocomposite films comprising a non-crosslinked, natural polymer (collagen) and a synthetic polymer, poly(var epsilon-caprolactone) (PCL), have been produced by impregnation of lyophilised collagen mats with a solution of PCL in dichloromethane followed by solvent evaporation. This approach avoids the toxicity problems associated with chemical crosslinking. Distinct changes in film morphology, from continuous surface coating to open porous format, were achieved by variation of processing parameters such as collagen:PCL ratio and the weight of the starting lyophilised collagen mat. Collagenase digestion indicated that the collagen content of 1:4 and 1:8 collagen:PCL biocomposites was almost totally accessible for enzymatic digestion indicating a high degree of collagen exposure for interaction with other ECM proteins or cells contacting the biomaterial surface. Much reduced collagen exposure (around 50%) was measured for the 1:20 collagen:PCL materials. These findings were consistent with the SEM examination of collagen:PCL biocomposites which revealed a highly porous morphology for the 1:4 and 1:8 blends but virtually complete coverage of the collagen component by PCL in the1:20 samples. Investigations of the attachment and spreading characteristics of human osteoblast (HOB) cells on PCL films and collagen:PCL materials respectively, indicated that HOB cells poorly recognised PCL but attachment and spreading were much improved on the biocomposites. The non-chemically crosslinked, collagen:PCL biocomposites described are expected to provide a useful addition to the range of biomaterials and matrix systems for tissue engineering.

Sustained delivery by leucine-modified chitosan spray-dried respirable powders

The controlled co-delivery of multiple agents to the lung offers potential benefits to patients. This study investigated the preparation and characterisation of highly respirable spray-dried powders displaying the sustained release of two chemically distinct therapeutic agents. Spray-dried powders were produced from 30% (v/v) aqueous ethanol formulations that contained hydrophilic (terbutaline sulphate) and hydrophobic (beclometasone dipropionate) model drugs, chitosan (as a drug release modifier) and leucine (aerosolisation enhancer). The influence of chitosan molecular weight on spray-drying thermal efficiency, aerosol performance and drug release profile was investigated. Resultant powders were physically characterised: with in vitro aerosolisation performance and drug release profile investigated by the Multi-Stage Liquid Impinger and modified USP II dissolution apparatus, respectively. It was found that increased chitosan molecular weight gave increased spray-drying thermal efficiency. The powders generated were of a suitable size for inhalation—with emitted doses over 90% and fine particle fractions up to 72% of the loaded dose. Sustained drug release profiles were observed in dissolution tests for both agents: increased chitosan molecular weight associated with increased duration of drug release. The controlled co-delivery of hydrophilic and hydrophobic entities underlines the capability of spray drying to produce respirable particles with sustained release for delivery to the lung. (c) 2009 Elsevier B.V. All rights reserved.

Modified release of beclometasone dipropionate from chitosan-basedd spray-dried respirable powders

Dry powders for inhalation were prepared by spray drying a 30% v/v aqueous ethanol formulation containing beclometasone dipropionate (BDP), lactose, leucine and chitosan (low, medium or high molecular weight (MW), or combinations thereof). Following physical characterisation of the powders, the aerosolisation and dissolution properties of the powders were investigated using Multi-Stage Liquid Impinger and USP II dissolution apparatus, respectively. The powders were highly dispersible, with emitted doses in excess of 90% of loaded powder aerosolised from a Spinhaler dry powder inhaler. The fine particle fraction (FPF) was observed to decrease, whereas the time for 100% drug release increased, with increasing chitosan MW. For example, the low MW formulation exhibited an FPF of 64% and a 100% dissolution time of 2 h, whereas the high MW formulation demonstrated an FPF of 54% and a dissolution time of 12 h. These powders would be anticipated to deposit predominately in the lower regions of the lung following inhalation, and then undergo delayed rather than instantaneous drug release, offering the potential to reduce dosing frequency and improve patient compliance. (c) 2008 Elsevier B.V. All rights reserved.

Chitosan-based spray-dried respirable powders for sustained delivery of terbutaline sulfate

In this study, we describe the preparation of highly dispersible dry powders for pulmonary drug delivery that display sustained drug release characteristics. Powders were prepared by spray-drying 30% v/v aqueous ethanol formulations containing terbutaline sulfate as a model drug, chitosan as a drug release modifier and leucine as an aerosolisation enhancer. The influence of chitosan molecular weight on the drug release profile was investigated by using low, medium and high molecular weight chitosan or combinations thereof. Following spray-drying, resultant powders were characterised using scanning electron microscopy, laser diffraction, tapped density analysis, differential scanning calorimetry and thermogravitational analysis. The in vitro aerosolisation performance and drug release profile were investigated using Multi-Stage Liquid Impinger analysis and modified USP II dissolution apparatus, respectively. The powders generated were of a suitable aerodynamic size for inhalation, had low moisture content and were amorphous in nature. The powders were highly dispersible, with emitted doses of over 90% and fine particle fractions of up to 82% of the total loaded dose, and mass median aerodynamic diameters of less than 2.5microm. A sustained drug release profile was observed during dissolution testing; increasing the molecular weight of the chitosan in the formulation increased the duration of drug release. (c)2007 Elsevier B.V. All rights reserved.

Tethering a laminin peptide to a crosslinked collagen scaffold for biofunctionality

Cell adhesion peptide regulates various cellular functions like proliferation, attachment, and spreading. The cellular response to laminin peptide (PPFLMLLKGSTR), a motif of laminin-5 alpha3 chain, tethered to type I collagen, crosslinked using microbial transglutaminase (mTGase) was investigated. mTGase is an enzyme that initiates crosslinking by reacting with the glutamine and lysine residues on the collagen fibers stabilizing the molecular structure. In this study that tethering of the laminin peptide in a mTGase crosslinked collagen scaffold enhanced cell proliferation and attachment. Laminin peptide tethered crosslinked scaffold showed unaltered cell morphology of 3T3 fibroblasts when compared with collagen and crosslinked scaffold. The triple helical structure of collagen remained unaltered by the addition of laminin peptide. In addition a dose-dependent affinity of the laminin peptide towards collagen was seen. The degree of crosslinking was measured by amino acid analysis, differential scanning calorimeter and fourier transform infrared spectroscopy. Increased crosslinking was observed in mTGase crosslinked group. mTGase crosslinking showed higher shrinkage temperature. There was alteration in the fibrillar architecture due to the crosslinking activity of mTGase. Hence, the use of enzyme-mediated linking shows promise in tethering cell adhesive peptides through biodegradable scaffolds.

Structural effects in photopolymerized sodium AMPS hydrogels crosslinked with poly(ethylene glycol) diacrylate for use as burn dressings

Synthetic hydrogel polymers were prepared by free radical photopolymerization in aqueous solution of the sodium salt of 2-acrylamido-2-methylpropane sulfonic acid (Na-AMPS). Poly(ethylene glycol) diacrylate (PEGDA) and 4,4'-azo-bis(4-cyanopentanoic acid) were used as the crosslinker and UV-photoinitiator, respectively. The effects of varying the Na-AMPS monomer concentration within the range of 30-50% w/v and the crosslinker concentration within the range of 0.1-1.0% mol (relative to monomer) were studied in terms of their influence on water absorption properties. The hydrogel sheets exhibited extremely high swelling capacities in aqueous media which were dependent on monomer concentration, crosslink density, and the ionic strength and composition of the immersion medium. The effects of varying the number-average molecular weight of the PEGDA crosslinker from = 250 to 700 were also investigated. Interestingly, it was found that increasing the molecular weight and therefore the crosslink length at constant crosslink density decreased both the rate of water absorption and the equilibrium water content. Cytotoxicity testing by the direct contact method with mouse fibroblast L929 cells indicated that the synthesized hydrogels were nontoxic. On the basis of these results, it is considered that photopolymerized Na-AMPS hydrogels crosslinked with PEGDA show considerable potential for biomedical use as dressings for partial thickness burns. This paper describes some structural effects which are relevant to their design as biomaterials for this particular application. © 2013 Copyright Taylor and Francis Group, LLC.

Evaluating transglutaminase crosslinked collagen gel systems for hard and soft tissue repair

Tissue Transglutaminase (TG2) and FXIIIa, members of the transglutaminase (TG) family, catalyses a transamidating reaction and form covalent bond between or within proteins. In bone development, both enzymes expressions correlate with the initial of the mineralisation process by osteoblasts and chondrocytes. Exogenous TG2 also promotes maturation of chondrocytes and mineralisation in pre-osteoblasts. To understand the role of endogenous TG2 in osteoblast mineralisation, the TG2 expression was examined during the human osteoblast (HOB) mineralisation. The expression of the endogenous TG2 increased during the mineralisation, yet, its expression was not essential for mineral deposition due to the compensation effect by other members in the TG family. The extracellular transamidating activity of HOBs was found increased during mineralisation and a shift from FXIIIa dominant- to TG2-dominant crosslinking activity was suggested after differentiation. However, the transamidating activity of both TG2 and FXIIIa were not critical for cell mineralisation. On the other hand, Exogenous TG2 was found to enhance wild type HOB and TG2 knockdown HOB mineral deposition. The transamidating activity of TG2 was not required but most likely a close conformation was essential for this enhancement. Results also demonstrated that exogenous TG2 may activate the ß-catenin pathway through LRP5 receptor thus contribute in cell mineralisation. This enhancement could be abolished by addition of ß-catenin inhibitors. Finally, using of TG2 crosslinked collagen gel for bone and cornea repair was evaluated. Crosslinked collagen gel showed promising results in improving HOB mineralisation, human corneal fibroblast (hCF) proliferation and migration. These effects might be resulted from the trapped TG2 within the collagen matrix and the alteration of matrix topography by TG2.

Chitosan-modified dry powder formulations for pulmonary gene delivery

Spray-drying is an effective process for preparing micron-dimensioned particles for pulmonary delivery. Previously, we have demonstrated enhanced dispersibility and fine particle fraction of spray-dried nonviral gene delivery formulations using amino acids or absorption enhancers as dispersibility-enhancing excipients. In this study, we investigate the use of the cationic polymer chitosan as a readily available and biocompatible dispersibility enhancer. Lactose-lipid:polycation:pDNA (LPD) powders were prepared by spray-drying and post-mixed with chitosan or spray-dried chitosan. In addition, the water-soluble chitosan derivative, trimethyl chitosan, was added to the lactose-LPD formulation before spray-drying. Spray-dried chitosan particles, displaying an irregular surface morphology and diameter of less than 2 mu m, readily adsorbed to lactose-LPD particles following mixing. In contrast with the smooth spherical surface of lactose-LPD particles, spray-dried trimethyl chitosan-lactose-LPD particles demonstrated increased surface roughness and a unimodal particle size distribution (mean diameter 3.4 mu m), compared with the multimodal distribution for unmodified lactose-LPD powders (mean diameter 23.7 mu m). The emitted dose and in vitro deposition of chitosan-modified powders was significantly greater than that of unmodified powders. Moreover, the inclusion of chitosan mediated an enhanced level of reporter gene expression. In summary, chitosan enhances the dispersibility and in vitro pulmonary deposition performance of spray-dried powders.

Stabilisation and characterisation of peroxide-crosslinked high density polyethylene

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Preparation and characterisation of novel polycaprolactone-chitosan blends

This research paper reports on the production of a biocompatible and biodegradable material to be used in a polymer stent used for counteracting the occurrence of anastomotic leakage following gastrointestinal surgery. Chitosan was blended with polycaprolactone in a solvent mixture of acetic acid and water. Membranes were formed with a range of 50/50%, 60/40%, 65/35%, 70/30% and 80/20% polycaprolactone/chitosan. The tensile properties of the blends were examined over a time period to access material degradation. In addition the biocompatibilities of the polycaprolactone/chitosan blends were tested for cytotoxic effect using primary tendon fibroblastic cells. This research concluded that the polycaprolactone/chitosan was non-toxic to the fibroblasts cells in-vitro. Analysis of the mechanical properties of the blends showed a range of mechanical strengths and polymer life spans. Overall, blends of 65/35%, 70/30% and 80/20% polycaprolactone/chitosan emerged as possible candidates for the production of a gastrointestinal stent. © 2011 Inderscience Enterprises Ltd.

Grafting functional antioxidants on highly crosslinked polyethylene

The problem of interference of antioxidants, such as hindered phenols, with peroxide-initiated crosslinking of polyethylene was addressed through the use of functional (reactive) graftable antioxidants (g-AO). Reactive derivatives of hindered phenol and hindered amine antioxidants were synthesised, characterised and used to investigate their grafting reactions in high density polyethylene; both non-crosslinked (PE) and highly peroxide-crosslinked (PEXa). Assessment of the extent of in-situ grafting of the antioxidants, their retention after exhaustive solvent extraction in PE and PEXa, and the stabilising performance of the grafted antioxidants (g-AO) in the polymer were examined and benchmarked against conventionally stabilised crosslinked & non-crosslinked polyethylene. It was shown that the functional antioxidants graft to a high extent in PEXa, and that the level of interference of the g-AOs with the polymer crosslinking process was minimal compared to that of conventional antioxidants which bear the same antioxidant function. The much higher level of retention of the g-AOs in PEXa after exhaustive solvent extraction, compared to that of the corresponding conventional antioxidants, accounts for their superior long-term thermal stabilising performance under severe extractive conditions.

Development and evaluation of a novel intranasal spray for the delivery of amantadine

The aim of this study was to develop and characterize an intranasal delivery system for amantadine hydrochloride (AMT). Optimal formulations consisted of a thermosensitive polymer Pluronic® 127 and either carboxymethyl cellulose or chitosan which demonstrated gel transition at nasal cavity temperatures (34 ± 1°C). Rheologically, the loss tangent (Tan δ) confirmed a 3-stage gelation phenomena at 34 ± 1°C and non-Newtonian behavior. Storage of optimized formulation carboxymethyl cellulose and optimal formulation chitosan at 4°C for 8 weeks resulted in repeatable release profiles at 34°C when sampled, with a Fickian mechanism earlier on but moving toward anomalous transport by week 8. Polymers (Pluronic® 127, carboxymethyl cellulose, and chitosan) demonstrated no significant cellular toxicity to human nasal epithelial cells up to 4 mg/mL and up to 1 mM for AMT (IC50: 4.5 ± 0.05 mM). Optimized formulation carboxymethyl cellulose and optimal formulation chitosan demonstrated slower release across an in vitro human nasal airway model (43%-44% vs 79 ± 4.58% for AMT). Using a human nasal cast model, deposition into the olfactory regions (potential nose-to-brain) was demonstrated on nozzle insertion (5 mm), whereas tilting of the head forward (15°) resulted in greater deposition in the bulk of the nasal cavity.

Administration routes affect the quality of immune responses:a cross-sectional evaluation of particulate antigen-delivery systems

Particulate delivery systems such as liposomes and polymeric nano- and microparticles are attracting great interest for developing new vaccines. Materials and formulation properties essential for this purpose have been extensively studied, but relatively little is known about the influence of the administration route of such delivery systems on the type and strength of immune response elicited. Thus, the present study aimed at elucidating the influence on the immune response when of immunising mice by different routes, such as the subcutaneous, intradermal, intramuscular, and intralymphatic routes with ovalbumin-loaded liposomes, N-trimethyl chitosan (TMC) nanoparticles, and poly(lactide-co-glycolide) (PLGA) microparticles, all with and without specifically selected immune-response modifiers. The results showed that the route of administration caused only minor differences in inducing an antibody response of the IgG1 subclass, and any such differences were abolished upon booster immunisation with the various adjuvanted and non-adjuvanted delivery systems. In contrast, the administration route strongly affected both the kinetics and magnitude of the IgG2a response. A single intralymphatic administration of all evaluated delivery systems induced a robust IgG2a response, whereas subcutaneous administration failed to elicit a substantial IgG2a response even after boosting, except with the adjuvanted nanoparticles. The intradermal and intramuscular routes generated intermediate IgG2a titers. The benefit of the intralymphatic administration route for eliciting a Th1-type response was confirmed in terms of IFN-gamma production of isolated and re-stimulated splenocytes from animals previously immunised with adjuvanted and non-adjuvanted liposomes as well as with adjuvanted microparticles. Altogether the results show that the IgG2a associated with Th1-type immune responses are sensitive to the route of administration, whereas IgG1 response associated with Th2-type immune responses were relatively insensitive to the administration route of the particulate delivery systems. The route of administration should therefore be considered when planning and interpreting pre-clinical research or development on vaccine delivery systems.

Novel hydrogel copolymers and semi-interpenetraing polymer networks

Interpenetrating polymer networks (lPN's), have been defined as a combination of two polymers each in network form, at least one of which has been synthesised and / or crosslinked in the presence of the other. A semi-lPN, is formed when only one of the polymers in the system is crosslinked, the other being linear. lPN's have potential advantages over homogeneous materials presently used in biomedical applications, in that their composite nature gives them a useful combination of properties. Such materials have potential uses in the biomedical field, specifically for use in hard tissue replacements, rigid gas permeable contact lenses and dental materials. Work on simply two or three component systems in both low water containing lPN's supplemented by the study of hydrogels (water swollen hydrophilic polymers) can provide information useful in the future development of more complex systems. A range of copolymers have been synthesised using a variety of methacrylates and acrylates. Hydrogels were obtained by the addition of N-vinyl pyrrolidone to these copolymers. A selection of interpenetrants were incorporated into the samples and their effect on the copolymer properties was investigated. By studying glass transition temperatures, mechanical, surface, water binding and oxygen permeability properties samples were assessed for their suitability for use as biomaterials. In addition copolymers containing tris-(trimethylsiloxy)-y-methacryloxypropyl silane, commonly abbreviated to 'TRlS', have been investigated. This material has been shown to enhance oxygen permeability, a desirable property when considering the design of contact lenses. However, 'TRIS' has a low polar component of surface free energy and hence low wettability. Copolymerisation with a range of methacrylates has shown that significant increases in surface wettability can be obtained without a detrimental effect on oxygen permeability. To further enhance to surface wettability 4-methacryloxyethyl trimellitic anhydride was incorporated into a range of promising samples. This study has shown that by careful choice of monomers it is possible to synthesise polymers that possess a range of properties desirable in biomedical applications.

Reactive processing of polyolefins using antioxidant systems

Various monoacrylic compounds containing a hindered phenol function (e.g.3,5-di-tert.-butyl-4-hydroxy benzyl alcohol, DBBA and vinyl-3-[3',5'-di-tert.-butyl-4-hydroxy phenyl] propionate, VDBP), and a benzophenone function (2-hydroxy-4-[beta hydroxy ethoxy] benzophenone, HAEB) were synthesised and used as reactive antioxidants (AO's) for polypropylene (PP). These compounds were reacted with PP melt in the presence of low concentration of a free radical generator such a peroxide (reactive processing) to produce bound-antioxidant concentrates. The binding reaction of these AO's onto PP was found to be low and this was shown to be mainly due to competing reactions such as homopolymerisation of the antioxidant. At high concentrations of peroxide, higher binding efficiency resulted, but, this was accompanied by melt degradation of the polymer. In a special reactive processing procedure, a di- or a trifunctional reactant (referred to as coagent), e.g.tri-methylol propane tri-acrylate, Tris, and Divinyl benzene, DVB, were used with the antioxidant and this has led to an enhanced efficiency of the grating reaction of antioxidants on the polymer in the melt. The evidence suggests that this is due to copolymerisation of the antioxidants with the coagent as well as grafting of the copolymers onto the polymer backbone. Although the 'bound' AO's containing a UV stabilising function showed lower overall stabilisation effect than the unbound analogues before extraction, they were still much more effective when subjected to exhaustive solvent extraction. Furthermore, a very effective synergistic stabilising activity when two reactive AO's containing thermal and UV stabilising functions e.g. DBBA and HAEB, were reactively processed with PP in the presence of a coagent. The stabilising effectiveness of such a synergist was much higher than that of the unbound analogues both before and after extraction. Analysis using the GPC technique of concentrates containing bound-DBBA processed in the presence of Tris coagent showed higher molecular weight (Mn), compared to that of a polymer processed without the coagent, but was still lower than that of the control processed PP with no additives. This indicates that Tris coagent may inhibit further melt degradation of the polymer. Model reactions of DBBA in liquid hydrocarbon (decalin) and analysis of the products using FTIR and NMR spectroscopy showed the formation of grafted DBBA onto decalin molecules as well as homopolymerisation of the AO. In the presence of Tris coagent, copolymerisation of DBBA with the Tris inevitably occured; which was followed by grafting of the copolymer onto the decalin, FTIR and NMR results of the polymer concentrates containing bound-DBBA processed with and without Tris, showed similar behaviour as the above model reactions. This evidence supports the effect of Tris in enhancing the efficiency of the reaction of DBBA in the polymer melt. Reactive procesing of HAEB in polymer melts exhibited crosslinking formation In the early stages of the reaction, however, in the final stage, the crosslinked structure was 'broken down' or rearranged to give an almost gel free polymer with high antioxidant binding efficiency.