Localized drug delivery using crosslinked gelatin gels containing liposomes: factors influencing liposome stability and drug release.

We describe a drug-delivery vehicle that combines the sustained release properties of liposomes with the structural advantages of crosslinked gelatin gels that can be implanted directly or coated onto medical devices. Liposome inclusion in gelatin gels does not compromise thermal stability nor does it interfere with the resiliency of gels to tensile force. However, electron spin resonance analysis of sequestered DPPC liposomes revealed a slight depression (ca. 1.0 degrees C) of the gel-to-fluid phase transition relative to liposomes in suspension. The level of liposome release from gels was determined by liposome concentration, liposome size, and the presence of poly(ethylene oxide) chains in the gel matrix or in the liposome membrane. Both neutral and charged liposomes displayed relatively high affinities for poly(ethylene glycol)gelatin gels, with only 10-15% release of initially sequestered liposomes while liposomes in which poly(ethylene glycol) was included within the membrane were not as well retained (approximately 65% release). The in vitro efflux of ciprofloxacin from liposomal gels immersed in serum was nearly complete after 24 h compared to 38% release of liposomal chlorhexidine after 6 days. The serum-induced destabilization of liposomal ciprofloxacin depended on the accessibility of serum components to gels as partly immersed gels retained approximately 50% of their load of drug after 24 h. In vivo experiments using a catheterized rabbit model of urinary tract infection revealed the absence of viable Escherichia coli on coated catheter surfaces in seven out of nine cases while all untreated catheter surfaces examined (n = 7) were contaminated.

Synergistic effect of active oxygen species and alginate on chitinase production by Wasabia japonica cells and its application.

The specific chitinase productivity of a Wasabia japonica cell suspension culture under pure oxygen aeration was 3.8 times higher than that of a suspension culture aerated with ordinary air. During aeration with pure oxygen, both oxygen consumption by the cells and the H2O2 concentration in the medium increased. Addition of H2O2 to the cultivation medium also promoted the specific chitinase productivity. H2O2 could pass freely through the cell membrane. It was assumed that the excess oxygen was converted into active oxygen species such as H2O2, and that the promotion of chitinase production was probably due to the generated active oxygen species. Addition of alginate oligomer (AO, an endogenous elicitor-like substance) to cultures aerated with pure oxygen or supplemented with H2O2 resulted in synergistic increases in chitinase production. Based on these results, the development of a simple and efficient chitinase production system was investigated. Cells were immobilized in alginate gel (instead of adding AO to the medium) and cultivated in a medium containing H2O2. The specific chitinase productivity increased to the levels observed in the suspension culture system. During repeated batch cultivation of immobilized cells, the chitinase production remained stable for three repeated batches. When immobilized protoplasts were cultivated in a medium containing H2O2, there was 7-fold increase in chitinase production compared with that of immobilized cells.

Antibiotic hydrogel coated Foley catheters for prevention of urinary tract infection in a rabbit model.

PURPOSE: We developed an antibiotic liposome (ciprofloxacin liposome) containing hydrogel for external coating of silicone Foley catheters and evaluated its efficacy in a rabbit model. Our goal was to create a catheter that would hinder the development of catheter associated nosocomial urinary tract infections. MATERIALS AND METHODS: We inserted either an untreated, liposomal hydrogel coated or a liposome hydrogel with ciprofloxacin coated 10F silicone Foley catheter into New Zealand White rabbits. We challenged the system with 5x10(6) virulent Escherichia coli at the urethral meatus twice daily for 3 days. Urine cultures were evaluated twice daily for 7 days. When urine cultures became positive, the rabbits were sacrificed and urine, urethral catheter and urethral tissue were cultured. RESULTS: The time to bacteriuria detection in 50% of the specimens was double for hydrogel with ciprofloxacin coated catheters versus untreated and hydrogel coated catheters. A significant (p = 0.04) improvement in average time to positive urine culture from 3.5 to 5.3 days and a 30% decrease in the bacteriuria rate for hydrogel with ciprofloxacin coated catheters were noted compared to untreated catheters. CONCLUSIONS: A significant benefit was realized by coating the extraluminal catheter surface with a ciprofloxacin liposome impregnated hydrogel. We believe this procedure will provide a significant clinical advantage, while reducing health care costs substantially.

A liposomal hydrogel for the prevention of bacterial adhesion to catheters.

The adhesion of bacteria to medical implants and the subsequent development of a biofilm frequently results in the infection of surrounding tissue and may require removal of the device. We have developed a liposomal hydrogel system that significantly reduces bacterial adhesion to silicone catheter material. The system consists of a poly (ethylene glycol)-gelatin hydrogel in which liposomes containing the antibiotic ciprofloxacin are sequestered. A poly (ethylene glycol)-gelatin-liposome mixture was applied to a silicone surface that had been pre-treated with phenylazido-modified gelatin. Hydrogel cross-linking and attachment to surface-immobilized gelatin was accomplished through the formation of urethane bonds between gelatin and nitrophenyl carbonate-activated poly (ethylene glycol). Liposomal hydrogel-coated catheters were shown to have an initial ciprofloxacin content of 185+/-16 microg cm(-2). Ciprofloxacin was released over seven days with an average release rate of 1.9+/-0.2 microg cm(-2) h(-1) for the first 94 h. In vitro assays using a clinical isolate of Pseudomonas aeruginosa established the antimicrobial efficacy of the liposomal hydrogel. A modified Kirby-Bauer assay produced growth-inhibition zone diameters of 39+/-1 mm, while bacterial adhesion was completely inhibited on catheter surfaces throughout a seven-day in vitro adhesion assay. This new antimicrobial coating shows promise as a prophylactic and/or treatment for catheter-related infection.

Purification and characterization of alpha-3',4'-anhydrovinblastine synthase (peroxidase-like) from Catharanthus roseus (L.) G. Don.

An H2O2-dependent enzyme capable of coupling catharanthine and vindoline into alpha-3',4'-anhydrovinblastine (AVLB) was purified to apparent homogeneity from Catharanthus roseus leaves. The enzyme shows a specific AVLB synthase activity of 1.8 nkat/mg, and a molecular weight of 45.40 kDa (SDS-PAGE). In addition to AVLB synthase activity, the purified enzyme shows peroxidase activity, and the VIS spectrum of the protein presents maxima at 404, 501 and 633 nm, indicating that it is a high spin ferric heme protein, belonging to the plant peroxidase superfamily. Kinetic studies revealed that both catharanthine and vindoline were substrates of the enzyme, AVLB being the major coupling product.

Molecular mobility of nitroxyl-labelled taxol during tubulin assembly.

Taxol is an important natural anticancer agent that binds to beta-tubulin and suppresses microtubule depolymerization. We have used electron paramagnetic resonance (EPR) spectroscopy to analyze the molecular motion of three novel nitroxyl free radical taxol analogues. Taxol was chemically modified at C2 or C7 carbon of the taxane ring with the TEMPO free radical to yield two spin-labelled taxols and concurrently at C2' and 3'N of the side chain to yield a spin-labelled taxol biradical. Nitroxyl moieties attached to the taxane ring are significantly restricted in their molecular motion during microtubule assembly, and they show no molecular restriction upon binding to tubulin. We conclude that taxol binds to tubulin in a way such that the taxane ring is not constrained by the dimer structure. However, the taxane ring is strongly immobilized after polymerization of tubulin, i.e. it is incorporated into the structure of microtubule. In contrast, the nitroxy moieties of the taxol biradical show strong immobilization upon attachment to tubulin. The nitroxyl energy exchange is restricted prior to the assembly of microtubules, and no differences associated with the process of polymerization were detected. The taxol side chain resides in a region that is not significantly constrained during polymerization.

Plant cell and tissue culture: alternatives for metabolite production.

Plant cell culture systems represent a potential renewable source of valuable medicinals, flavours, essences and colourants that cannot be produced by microbial cells or chemical syntheses. However, only a few cultures produce these compounds in commercially useful amounts. The low productivities are associated with our poor understanding of the biochemistry of these systems. Recent advances in molecular biology, enzymology, physiology and fermentation technology of plant cell cultures suggest that these systems will become a viable source of important natural products. This review examines the sate of the art of production of medicinal plant secondary metabolites by plant cell cultures.

Improved taxol yield by aromatic carboxylic acid and amino acid feeding to cell cultures of taxus cuspidata.

Cell culture of Taxus cuspidata represents an alternative to whole plant extraction as a source of taxol and related taxanes. Feeding phenylalanine to callus cultures was previously shown to result in increased taxol yields, probably due to the involvement of this amino acid as a precursor for the N-benzoylphenylisoserine side chain of taxol. Inthis study, we have examined the effect of various concentrations of phenylalanine, benzoic acid, N-benzoylglycine, serine, glycine, alanine, and 3-amino-3-phenyl-propionic acid on taxol accumulation in 2-year-old cell suspensions of Taxus cuspidata, cell line FCL1F, and in developing callus cultures of T. cuspidata. All compounds tested were included in media at stationary phase (suspensions) or after the period of fastest growth (calli). Alanine and 3-amino-3-phenyl-propionicacid were tested only in callus cultures and did not affect taxol accumulation. Significant increases or trends toward increases in taxol accumulationin callus and suspensions were observed in the presence of phenylalanine, benzoic acid, N-benzoylglycine, serine, and glycine. The greatest increases in taxol accumulation were observed in the presence of various concentrations of phenylalanine (1 mM for callus; 0.05, 0.1, and 0.2 mM for suspensions) and benzoic acid (0.2 and 1 mM for callus and 0.05, 0.1, and 0.2 mM for suspensions). Increases in taxol yields of cell suspensions in the presence of the most effective precursors brought taxol amounts at stationary phase from 2 mug . g(-1) to approximately 10 mug . g(-1) of the extracted dry weight. The results are discussed in termsof possible implications to taxol biosynthesis and in terms of practical applications to large-scale cell culture systems for the production ofthis drug. (c) 1994 John Wiley & Sons, Inc.

Kinetics of taxol production, growth, and nutrient uptake in cell suspensions of Taxus cuspidata.

Cell culture of Taxus cuspidata may represent an alternative to extraction of bark as a source of taxol and related taxanes. Cell suspensions of a cell line of T. cuspidata were grown for 44 days in shake flasks containing B5C2 medium. Throughout the growth cycle, fresh and dry weight accumulation, taxol yield on a dry weight basis, taxol accumulation in the medium, pH and pigmentation variation in the medium, as well as the uptake of sucrose, glucose, fructose, nitrate, and inorganic phosphate from the culture medium were examined. The results showed that the growth was relatively slow (doubling times of 17 and 20 days for fresh and dry weight, respectively), and taxol accumulation in the cells was non-growth related (higher in the stationary phase) and at relatively low levels (up to 4 mug/g of the extracted dry weight). Taxol concentration in the medium had two peaks: one during the early (0.4mug/mL) and another during the late (0.1-mug/mL) parts of the growth cycle. On a volumetric basis, the average total amount of taxol produced during the stationary phase (day 38) was 0.15 mug/mL, of which approximately 66% was in the medium and 34% was in the cells. Total carbohydrate uptake was closely associated with the increase in dry biomass. Sucrose was apparently extracellularly hydrolyzed after the first 6 days of culture; glucose was used before fructose. Nitrate was assimilated throughout the growth cycle, but phosphate was absorbed within the first week of culture. The pH variation showed an initial drop followed by a trend toward alkalinization for most of the growth period. Dark pigmentation in the medium increased progressively, particularly during the stationary phase. (c) 1994 John Wiley & Sons, Inc.

Improved growth and taxol yield in developing calli of Taxus cuspidata by medium composition modification.

Cell culture of Taxus spp. represents a potential alternative source of taxol and related taxanes used in cancer chemotherapy. We have analyzed the effect of different culture media components on growth and production of taxol in developing callus cultures of T. cuspidata. Several sequential modifications were made to the basal B5 medium, which included addition and/or variation in the concentration of sucrose, B5 organic supplements, gibberellic acid, 36 combinations of 2,4-D/kinetin ratios, media salts and organic supplements, phenylalanine, casein hydrolysate and medium pH. The experiments were conducted during a 55 day-growth period followed by taxane extraction and analysis. Significant increases in taxol yield and growth over basal medium grown calli were observed with some of the modified media.

Cell culture of Taxus as a source of the antineoplastic drug taxol and related taxanes.

Callus cultures of Taxus cuspidata and Taxus canadensis were induced using different tissue explants including green and red arils, seed contents, young stems and needles. Callus derived from stem segments displayed the best growth in defined media. The culture medium was supplemented with reducing agents and phenolic-binding compounds to inhibit callus darkening and subsequent growth reduction. T. cuspidata explant growth was affected by different concentrations and ratios of 2,4-D and kinetin. Callus tissues of T. cuspidata were extracted for taxol after 2 months in culture and analysed by HPLC. The presence of taxol (0.020 +/- 0.005% of the extracted dry weight) was indicated based on retention time, U.V. spectra, peak purity as assessed by photo-diode array spectroscopy and compared with an authentic taxol standard, as well as by 1H-NMR analysis. Suspension cultures of T. cuspidata were established from the callus cultures, subsequently immobilized onto glass fiber mats, and maintained as immobilized cultures for 6 months. The immobilized cell cultures also produced taxol at levels up to 0.012 +/- 0.007% of the extracted dry weight.

Distribution and amounts of taxol in different shoot parts of Taxus cuspidata.

Different fresh shoot parts of male and female plants of Taxus cuspidata were extracted and analysed for taxol concentration by high performance liquid chromatography (HPLC). Extracted parts included: young needles (first 10 top needle pairs of 30 cm long branches), old needles (last 10 needle pairs of 30 cm long branches), green bark, dark bark (with intense secondary growth), young wood (originally surrounded by green bark), wood (originally surrounded by dark bark), young stems (surrounded by green bark and devoid of needles), and mature male cones. Dichloromethane extracts were analysed by HPLC and diode array spectroscopy. Taxol identification was done by retention time, U.V. spectra, and spiking with an authentic taxol standard; 1H-NMR analysis was done for needle extracts. All parts except male cones had measurable amounts of taxol; no effect of plant sex on taxol levels of the plant parts analysed was observed. Results indicated that the bark accounted for almost all the taxol present in stems devoid of needles. Needles showed the highest levels of taxol (overall average of 0.035 +/- 0.006% of the extracted dry weight), significantly higher than those displayed by dark bark samples (0.012 +/- 0.001% of the extracted dry weight). Different needle post-harvesting procedures were evaluated in relation to taxol yields, 96 h dark incubation at -12 degrees C and 96 h dark incubation at 25 degrees C under vacuum gave taxol yields equivalent to those of freshly extracted samples. However, results obtained for 96 h dark incubation at 60 degrees C indicated some extent of taxol degradation.

Secondary metabolite biosynthesis in cultured cells of Catharanthus roseus (L.) G. Don immobilized by adhesion to glass fibres.

Suspension-cultured cells of Catharanthus roseus (L.) G. Don were immobilized on glass fibre mats and cultivated in shake flasks. The highly-aggregated immobilized cells exhibited a slower growth rate and accumulated reduced levels of tryptamine and indole alkaloids, represented by catharanthine and ajmalicine, in comparison to cells in suspension. The increased total protein synthesis in immobilized cells suggests a diversion of the primary metabolic flux toward protein biosynthetic pathways and away from other growth processes. In-vitro assays for the specific activity of tryptophan decarboxylase (TDC) and tryptophan synthase (TS) suggest that the decreased accumulation of tryptamine in immobilized cells was due to reduced tryptophan biosynthesis. The specific activity of TDC was similar in immobilized and suspension-cultured cells. However, the expression of TS activity in immobilized cells was reduced to less than 25% of the maximum level in suspension-cultured cells. The reduced availability of a free tryptophan pool in immobilized cells is consistent with the reduced TS activity. Reduced tryptamine accumulation, however, was not responsible for the decreased accumulation of indole alkaloids in immobilized cells. Indole alkaloid accumulation increased to a similar level in immobilized and suspension-cultured cells only after the addition of exogenous secologanin to the culture medium. The addition of tryptophan resulted in increased accumulation of tryptamine, but had no effect on indole alkaloid levels. Reduced biosynthesis of secologanin, the monoterpenoid precursor to indole alkaloids, in immobilized cells is suggested. Immobilization does not appear to alter the activity of indole alkaloid biosynthetic enzymes in our system beyond, and including, strictosidine synthase.

Plant cell bioreactor for the production of protoberberine alkaloids from immobilized Thalictrum rugosum cultures.

Cultured Thalictrum rugosum cells were immobilized using a glass fiber substratum previously shown to provide optimum immobilization efficiency based on spontaneous adhesion mechanisms. When cultivated in shake flasks, immobilized cells exhibited decreased growth and protoberberine alkaloid production rates in comparison to freely suspended cells. Since alkaloid production is growth associated in T. rugosum, the decreased specific production rate was a function of the slower growth rate. Cells immobilized on glass fiber mats appear to be amenable for extended culture periods. Maximum biomass and protoberberine alkaloid levels were maintained for at least 14 days in immobilized cultures. In contrast, fresh weight, dry weight, and total alkaloid content decreased in suspension cultures following the linear growth phase. Glass fiber mats were incorporated in to a 4.5-L plant cell bioreactor as horizontal disks supported on a central rod. Mixing in the reactor was provided by the combined actions of a magnetic impeller and a cylindrical sparging column. The magnetic impeller and a cylindrical sparging column. The entire inoculum biomass of T. rougosum, introduced as suspension, was spontaneously immobilized with in 8 h. During liner phase, the growth rate of bioreactor cultivated immobilized cells (mu = 0.06 day(-1)) was 50% that immobilized cell viability in both mmobilized cells cultivated in shake flasks (mu = 0.12 day(-1)). Percentage of cell viability in both systems was determined to be similar. The growing biomass in the bioreactor was completely retained by the glass fiber substratum during a 28-day culture period. The increase in specific production of protoberberine alkaloids was initially similar in bioreactor- and shake-flask-cultivated immobilized cells. However, the maximum specific production of bioreactor-grown cultures was lower. The scaleup potential of an immobilization strategy based on the spontaneous adhesion of cultured plant cells to glass fibers is demonstrated.

Immobilization of cells by spontaneous adhesio.

Various immobilization strategies have been developed to optimize the biosynthetic potential of cultured plant cells. Immobilization involves the retaining of suspension-cultured plant cells on, or within, a physical barrier that promotes cell aggregation and separates the cells from the surrounding media. The advantages of immobilization include the formation of diffusionary gradients around and between the cells that increase intercellular biochemical communication conducive to the coordinated expression of secondary metabolism. In effect, immobilization simulates the physiological conditions within large aggregates of cells in a manner amenable to manipulation for fermentation purposes.

Adhesion of suspension-cultured Catharanthus roseus cells to surfaces: effect of pH, ionic strength, and cation valency.

The correlation between the effects of pH, ionic strength and cation valency on the electrophoretic mobility and the extent of adhesion of suspension-cultured Catharanthus roseus cells to various polymer substrates is presented. The electrophoretic mobility of cells was unaltered in the pH range of 6-8, but decreased from approximately -2.2 x 10(-8) m V-1 s-1 and approached zero as the pH of the suspending liquid was decreased from 6 to 2. Similarly, the value of electrophoretic mobility decreased continuously as the ionic strength was increased from 0 to 1.0 M when cells were suspended in salt solutions of sodium chloride, calcium chloride, and aluminum chloride. However, using equimolar concentrations, the slope of the decrease in electrophoretic mobility increased following the sequence sodium chloride less than calcium chloride less than aluminium chloride. The electrophoretic mobility was near zero for suspensions containing 1.0 M calcium chloride or 0.1 M aluminium chloride. The extent of adhesion of the cells to the polymers sulphonated polystyrene less than polyethylene terephthalate less than polystyrene less than fluorinated ethylene-propylene followed this sequence. These results agree with a thermodynamic model of plant cell adhesion that implicates the importance of interfacial tensions in the adhesion process. However, higher levels of adhesion were generally observed when the electrophoretic mobility for the cells in the corresponding test liquid was at a minimum absolute value. These results can be explained by considering the effects of the electrolytic properties of the suspending liquid on the electrostatic repulsive interactions between the cells and the polymer surface in terms of a double-layer phenomenon and the DLVO theory.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of Indole Alkaloid Content in Vanadium-Treated Catharanthus roseus Suspension Cultures.

Addition of vanadyl sulphate to cell suspensions of CATHARANTHUS ROSEUS was found to increase ajmalicine, catharanthine, and tryptamine levels. Up to 500 microg/g dry weight catharanthine and 131.0 microg/g dry weight ajmalicine were detected in vanadyl sulphate-treated cells. This represents an approximate increase of 50% over control levels. This stimulation was found to be dependent upon the concentration of vanadyl sulphate administered and upon the cell age. High tryptamine levels were not correlated with increased tryptophan decarboxylase activity. Vanadium content of the cells was found to reach a maximum within 1 min following vanadyl sulphate treatment as measured by Instrumental Neutron Activation Analysis.

Increased synthesis of ajmalicine and catharanthine by cell suspension cultures of Catharanthus roseus in response to fungal culture-filtrates.

The ammonium sulfate-precipitated fraction from mycelia and culture-filtrates and the crude, cell-free culture filtrates from the growth medium of the fungi Chrysosporium palmorum, Eurotium rubrum, Micromucor isabellina, and Pythium aphanidermatum when aseptically added to cell suspensions of Cantharanthus roseus caused a rapid and dramatic increase in indole alkaloid biosynthesis. Up to 400 micrograms/L ajmalicine and 600 micrograms/L catharanthine were detected in C. roseus cell suspension grown in the presence of the M. isabellina fungal culture filtrate for 3 d. Untreated cells produced only trace levels of ajmalicine and catharanthine per liter of cell suspension after 15 d of culture.

Increased accumulation of indole alkaloids by some cell lines of Catharanthus roseus in response to addition of vanadyl sulphate.

Vanadyl sulphate (10-500 mg/l), when added to cell suspension cultures of Catharanthus roseus stimulated increased intracellular accumulation of catharanthine and ajmalicine. This response was demonstrated in both flask and fermenter (30 litre) systems. The response varied, and depended upon cell line, concentration of vanadyl sulphate and the stage of the growth phase at which the cells were treated. This process has the potential to increase the yield and reduce the production time for commercially useful secondary plant metabolites.