Controlled release of an antibiotic, gentamicin sulphate, from gravity spun polycaprolactone fibers.

The antibiotic, gentamicin sulphate (GS), was incorporated in gravity-spun polycaprolactone (PCL) fibers by spinning from particulate suspensions of the drug in PCL solution to produce a controlled delivery system. The production rate of GS-loaded PCL fibers was confined to the range 1-1.5 m/min and the fiber diameter to 170-220 microm. The kinetics of drug release could be adjusted by varying the GS loading of the fibers and the suspension preparation conditions. Gradual release of approximately 80% of the initial GS content was measured in phosphate buffered saline at 37 degrees C over 50 days from fibers spun from nonhomogenized suspensions, whereas loss of this amount of antibiotic occurred in less than 10 days from fibers spun from homogenized suspensions. Studies of growth inhibition of Stapyhlococcus epidermidis in culture indicated that GS released after 2 weeks from PCL fibers retained antibacterial activity. This behavior recommends further investigation of PCL fibers for local delivery of antibiotics to combat infection associated with periodontal disease, musculoskeletal injuries, and implantation of fiber-based tissue substitutes such as vascular prostheses.

Delivery of the antibiotic gentamicin sulphate from precipitation cast matrices of polycaprolactone.

Microporous, poly(epsilon-caprolactone) (PCL) matrices were loaded with the aminoglycoside antibiotic, gentamicin sulphate (GS) using the precipitation casting technique by suspension of powder in the PCL solution prior to casting. Improvements in drug loading from 1.8% to 6.7% w/w and distribution in the matrices were obtained by pre-cooling the suspension to 4 degrees C. Gradual release of approximately 80% of the GS content occurred over 11 weeks in PBS at 37 degrees C and low amounts of antibiotic were measured up to 20 weeks. The kinetics of release could be described effectively by the Higuchi model with the diffusion rate constant (D) increasing from of 1.7 to 5.1 microg/mg matrix/day(0.5) as the drug loading increased from 1.4% to 8.3% w/w. GS-loaded PCL matrices retained anti-bacterial activity after immersion in PBS at 37 degrees C over 14 days as demonstrated by inhibition of growth of S. epidermidis in culture. These findings recommend further investigation of precipitation-cast PCL matrices for delivery of hydrophilic molecules such as anti-bacterial agents from implanted, inserted or topical devices.

Precipitation casting of drug-loaded microporous PCL matrices: incorporation of progesterone by co-dissolution.

Microporous, poly(epsilon-caprolactone) (PCL) matrices were loaded with progesterone by precipitation casting using co-solutions of PCL and progesterone in acetone. Progesterone loadings up to 32% w/w were readily achieved by increasing the drug content of the starting PCL solution. The kinetics of steroid release in PBS at 37 degrees C over 10 days could be described effectively by a diffusional release model although the Korsmeyer-Peppas model indicated the involvement of multiple release phenomena. The diffusion rate constant (D) increased from 8 to 24 microg/mg matrix/day0.5 as the drug loading increased from 3.6 to 12.4% w/w. A total cumulative release of 75%-95% indicates the high efficiency of steroid delivery. Increasing the matrix density from 0.22 to 0.39 g/cm3, by increasing the starting PCL solution concentration, was less effective in changing drug release kinetics. Retention of anti-proliferative activity of released steroid was confirmed using cultures of breast cancer epithelial (MCF-7) cells. Progesterone released from PCL matrices into PBS at 37 degrees C over 14 days retarded the growth of MCF-7 cells by a factor of at least 3.5 compared with progesterone-free controls. These findings recommend further investigation of precipitation-cast PCL matrices for delivery of bioactive molecules such as anti-proliferative agents from implanted, inserted or topical devices.

A co-cultured skin model based on cell support membranes.

Tissue engineering of skin based on collagen:PCL biocomposites using a designed co-culture system is reported. The collagen:PCL biocomposites having collagen:PCL (w/w) ratios of 1:4, 1:8, and 1:20 have been proven to be biocompatible materials to support both adult normal human epidermal Keratinocyte (NHEK) and mouse 3T3 fibroblast growth in cell culture, respectively, by Dai, Coombes, et al. in 2004. Films of collagen:PCL biocomposites were prepared using non-crosslinking method by impregnation of lyophilized collagen mats with PCL/dichloromethane solutions followed by solvent evaporation. To mimic the dermal/epidermal structure of skin, the 1:20 collagen:PCL biocomposites were selected for a feasibility study of a designed co-culture technique that would subsequently be used for preparing fibroblast/biocomposite/keratinocyte skin models. A 55.3% increase in cell number was measured in the designed co-culture system when fibroblasts were seeded on both sides of a biocomposite film compared with cell culture on one surface of the biocomposite in the feasibility study. The co-culture of human keratinocytes and 3T3 fibroblasts on each side of the membrane was therefore studied using the same co-culture system by growing keratinocytes on the top surface of membrane for 3 days and 3T3 fibroblasts underneath the membrane for 6 days. Scanning electron microscopy (SEM) and immunohistochemistry assay revealed good cell attachment and proliferation of both human keratinocytes and 3T3 fibroblasts with these two types of cells isolated well on each side of the membrane. Using a modified co-culture technique, a co-cultured skin model presenting a confluent epidermal sheet on one side of the biocomposite film and fibroblasts populated on the other side of the film was developed successfully in co-culture system for 28 days under investigations by SEM and immunohistochemistry assay. Thus, the design of a co-culture system based on 1:20 (w/w) collagen:PCL biocomposite membranes for preparation of a bi-layered skin model with differentiated epidermal sheet was proven in principle. The approach to skin modeling reported here may find application in tissue engineering and screening of new pharmaceuticals.

Improving peptide-based assays to differentiate between vaccination and Mycobacterium bovis infection in cattle using nanoparticle carriers for adsorbed antigens.

The development of diagnostic tests to differentiate between vaccinated animals and those infected with Mycobacterium bovis is required so that test and slaughter control strategies can continue alongside vaccination. In this work, the peptide antigen, ESAT-6, p45, derived from the N-terminal sequence of the ESAT-6 protein, was adsorbed onto a range of microparticulate and nanoparticulate substrates to enhance the in vitro immune response of blood lymphocytes previously sensitised to M. bovis. Two types of hydroxyapatite (HA) nanoparticles (both approximately 300 nm in linear dimension), carbonate hydroxyapatite nanospheres (CHA, approximately 50 nm), two sizes of polystyrene nanospheres ( approximately 500 and 40 nm), calcium carbonate microparticles (0.3-1.0 microm) and glass microspheres (1.0-3.0 microm) were incubated in a solution of the peptide in PBS. Peptide adsorption increased on the nanoparticle carriers in the order HA (2.5+/-0.12%w/w), CHA (4.9+/-0.12) polystyrene (500 nm, 6.8+/-0.15%, 40 nm, 9.2+/-0.07) and these systems exhibited fairly low levels of desorption (approximately 10-15% peptide release) over a 24-h incubation period in PBS at 37 degrees C. HA, CHA and polystyrene carriers with adsorbed peptide were subsequently tested in the BOVIGAM assay to investigate the efficiency of the immune response of blood lymphocytes in terms of interferon-gamma (IFN-gamma) production. A general elevation of IFN-gamma production resulted for particle-bound peptide relative to free peptide at high peptide concentrations (>10 microg/ml). Only HA-adsorbed peptide resulted in consistently higher immune responses at low peptide concentration (<0.1 microg/ml) compared with the free peptide, indicating that peptide antigens adsorbed to hydroxyapatite nanoparticles may be useful, in diagnostic assays, for differentiating between tuberculosis (TB)-infected and vaccinated animals.

Liposome formulation of poorly water soluble drugs: optimisation of drug loading and ESEM analysis of stability.

Liposomes due to their biphasic characteristic and diversity in design, composition and construction, offer a dynamic and adaptable technology for enhancing drug solubility. Starting with equimolar egg-phosphatidylcholine (PC)/cholesterol liposomes, the influence of the liposomal composition and surface charge on the incorporation and retention of a model poorly water soluble drug, ibuprofen was investigated. Both the incorporation and the release of ibuprofen were influenced by the lipid composition of the multi-lamellar vesicles (MLV) with inclusion of the long alkyl chain lipid (dilignoceroyl phosphatidylcholine (C24PC)) resulting in enhanced ibuprofen incorporation efficiency and retention. The cholesterol content of the liposome bilayer was also shown to influence ibuprofen incorporation with maximum ibuprofen incorporation efficiency achieved when 4 micromol of cholesterol was present in the MLV formulation. Addition of anionic lipid dicetylphosphate (DCP) reduced ibuprofen drug loading presumably due to electrostatic repulsive forces between the carboxyl group of ibuprofen and the anionic head-group of DCP. In contrast, the addition of 2 micromol of the cationic lipid stearylamine (SA) to the liposome formulation (PC:Chol - 16 micromol:4 micromol) increased ibuprofen incorporation efficiency by approximately 8%. However further increases of the SA content to 4 micromol and above reduced incorporation by almost 50% compared to liposome formulations excluding the cationic lipid. Environmental scanning electron microscopy (ESEM) was used to dynamically follow the changes in liposome morphology during dehydration to provide an alternative assay of liposome stability. ESEM analysis clearly demonstrated that ibuprofen incorporation improved the stability of PC:Chol liposomes as evidenced by an increased resistance to coalescence during dehydration. These finding suggest a positive interaction between amphiphilic ibuprofen molecules and the bilayer structure of the liposome.

Measuring the heterogeneity of protein loading in PLG microspheres using flow cytometry.

Poly (DL-lactide co-glycolide) (PLG) microspheres with mean sizes up to 1 microm containing Fluorescein Isothiocyanate labelled Bovine Serum Albumin (FITC-BSA) were prepared by the water-in oil-in water (w/o/w) emulsion solvent evaporation technique. Protein loading and loading efficiency determined by the BCA total protein assay increased with microsphere size as measured by laser diffractometry. Protein loaded microspheres were analysed using flow cytometry (FC) to provide fast and reproducible measurements of the size and protein loading of individual microspheres within a sample thereby quantifying in detail the batch heterogeneity. The FC analysis demonstrated that as the size of individual microspheres within a batch increased, so the protein loading tended to increase. For example, the protein loading of microspheres increased from 2.7 to 8.9 wt.% as the size of microspheres increased from 0.42 to 1.45 microm, respectively. Measurements taken during a subsequent protein release experiment indicated that smaller microspheres within a sample released their protein more quickly than larger sizes. Flow cytometry has been shown to provide detailed information, at the level of individual microspheres, about the heterogeneity in size and protein loading of a microsphere sample and could thus lead to improvement of the release characteristics of microsphere-based delivery systems for biopharmaceuticals.

Composite cell support membranes based on collagen and polycaprolactone for tissue engineering of skin.

The preparation and characterisation of collagen:PCL composites for manufacture of tissue engineered skin substitutes and models are reported. Films having collagen:PCL (w/w) ratios of 1:4, 1:8 and 1:20 were prepared by impregnation of lyophilised collagen mats by PCL solutions followed by solvent evaporation. In vitro assays of collagen release and residual collagen content revealed an expected inverse relationship between the collagen release rate and the content of synthetic polymer in the composite that may be exploited for controlled presentation and release of biopharmaceuticals such as growth factors. DSC analysis revealed the characteristic melting point of PCL at around 60 degrees C and a tendency for the collagen component, at high loading, to impede crystallinity development within the PCL phase. The preparation of fibroblast/composite constructs was investigated using cell culture as a first stage in mimicking the dermal/epidermal structure of skin. Fibroblasts were found to attach and proliferate on all the composites investigated reaching a maximum of 2 x 10(5)/cm(2) on 1:20 collagen:PCL materials at day 8 with cell numbers declining thereafter. Keratinocyte growth rates were similar on all types of collagen:PCL materials investigated reaching a maximum of 6.6 x 10(4)/cm(2) at day 6. The results revealed that composite films of collagen and PCL are favourable substrates for growth of fibroblasts and keratinocytes and may find utility for skin repair.

Precipitation casting of polycaprolactone for applications in tissue engineering and drug delivery.

Microporous materials have been produced by gradual precipitation from solutions of poly(epsilon-caprolactone) (PCL) in acetone induced by solvent extraction across a semi-permeable PCL membrane which is formed in situ at the polymer solution/non-solvent interface. Microparticulates of hydroxyapatite and inulin polysaccharide, respectively, were incorporated in precipitation cast PCL matrices to illustrate potential applications in hard tissue repair and macromolecular drug release. Microporous PCL and HA filled PCL materials were found to provide a favourable surface for attachment and growth of primary human osteoblasts in cell culture. The in vitro degradation characteristics of microporous PCL and inulin/PCL materials in PBS at 37 degrees C were monitored over 45 months. Microporous PCL demonstrated zero weight loss, minor changes in molecular weight characteristics and a fairly constant indentation resistance of around 1 MN/m2. Inulin-loaded PCL materials exhibited a total weight loss of approximately 17% after 12 months in PBS. The indentation resistance decreased by 50% from an initial value of 28 MN/m2 in the first 2 months and then remained stable. Precipitation cast materials based on PCL are expected to be useful for formulating long-term, controlled release devices for bioactive molecules such as growth factors and hormones and extended-residence supports for cell growth and tissue development.

Japanese encephalitis virus vaccine formulations using PLA lamellar and PLG microparticles.

Japanese encephalitis virus (JEV)-loaded poly(lactide) (PLA) lamellar and poly(DL-lactide-co-glycolide) (PLG) microparticles were successfully prepared with low molecular weight PLA by the precipitate method and with 6% w/v PLG in the organic phase, 10% w/v PVP and 5% w/v NaCl in the continuous phase, by using a water-in-oil-in-water emulsion/solvent extraction technique, respectively. JEV was entrapped in the PLG microparticles by a solvent extraction technique with trapping efficiencies up to 98%, loading level 5.5% w/w, and mean particle size 3.8 microm. The distribution (%) of JEV on the PLG microparticles surface, outer layer, and core were 11.2, 41.7 and 46.4%, respectively. The cumulative release of JEV had an upper limit of approximately 58% of the JEV load at 24 days. The steady release rate was 1.33 microg JEV/mg microparticles/day of JEV release maintained for 24 days. The corresponding virus loading of the PLA lamellae is approximately 0.78% w/w and the loading efficiency (77.8%), JEV content (7.84 microg/mg), and yield (96.3%), respectively. The distribution (%) of JEV on the microparticles surface, outer layer, and core were 82.1, 13.3 and 2.2%, respectively. The live JEV challenge in mice test, in which mice received one dose of 20 mg JEV-loaded PLG microparticles, 20 mg JEV-loaded PLA lamellar in comparison with JEV or PBS solution, was evaluated after IP immunization of BALB/c mice. The study results show that the greatest survival was observed in the group of mice immunized with 20 mg JEV-loaded PLG microparticles and 20 mg JEV-loaded PLA microparticles group (80%). The JEV incorporation, physicochemical characterization data, and the animal results obtained in this study may be relevant in optimizing the vaccine incorporation and delivery properties of these potential vaccine targeting carriers.

Biocomposites of non-crosslinked natural and synthetic polymers.

Biocomposite films comprising a non-crosslinked, natural polymer (collagen) and a synthetic polymer, poly(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 the 1:20 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.

In vitro behavior of albumin-loaded carbonate hydroxyapatite gel.

Hydroxyapatite (HA) powder, porous HA, plasma-sprayed HA, apatite cements, and sintered HA have all been investigated as delivery systems for compounds such as human growth hormone and vancomycin. However, many previous studies showed that the period of release was limited to 2-3 weeks. The concept of using a nanoporous matrix as a means of immobilizing proteins is well known but has largely been confined to silica-based systems. Carbonate hydroxyapatite (CHA) is more soluble in vivo than HA, and when formed as an aqueous precipitate, it is often formed as nanocrystals. This study investigated the release profiles of ovine albumin (OVA) from CHA gel stored in phosphate-buffered saline (PBS) and double distilled water (DDW) for times of up to 1 year. It was found that 7.9% OVA could be loaded onto apatitic gels by means of a purely aqueous process. This process provided a simple low-temperature method of protein adsorption on a high surface area apatitic matrix at physiological pH. The rate of short-term release of OVA was lower from CHA gels than from microcrystalline HA powder. However, the period of release from the CHA gel was short term and may have been associated with recrystallization of the gel. OVA loaded into CHA gel was found to remain undegraded in vitro at 37 degrees C for periods of up to 1 year.