Lamellar crystalline self-assembly behaviour and solid lipid nanoparticles of a palmityl prodrug analogue of Capecitabine--a chemotherapy agent.

An amphiphile prodrug, 5'-deoxy-5-fluoro-N(4)-(palmityloxycarbonyl) cytidine or 5'-deoxy-5-fluoro-N(4)-(hexadecanaloxycarbonyl) cytidine (5-FCPal), consisting of the same head group as the commercially available chemotherapeutic agent Capecitabine, linked to a palmityl hydrocarbon chain via a carbamate bond is reported. Thermal analysis of this prodrug indicates that it melts at ∼115 °C followed quickly by degradation beginning at ∼120 °C. The neat solid 5-FCPal amphiphile acquires a lamellar crystalline arrangement with a d-spacing of 28.6±0.3 Å, indicating interdigitation of the hydrocarbon chains. Under aqueous conditions, solid 5-FCPal is non-swelling and no lyotropic liquid crystalline phase formation is observed. In order to assess the in vitro toxicity and in vivo efficacy in colloidal form, solid lipid nanoparticles (SLNs) with an average size of ∼700 nm were produced via high pressure homogenization. The in vitro toxicity of the 5-FCPal SLNs against several different cancer and normal cell types was assessed over a 48 h period, and IC(50) values were comparable to those observed for Capecitabine. The in vivo efficacy of the 5-FCPal SLNs was then assessed against the highly aggressive mouse 4T1 breast cancer model. To do so, the prodrug SLNs were administered orally at 3 different dosages (0.1, 0.25, 0.5 mmol/mouse/day) and compared to Capecitabine delivered at the same dosages. After 21 days of receiving the treatments, the 0.5 mmol dose of 5-FCPal exhibited the smallest average tumour volume. Since 5-FCPal is activated in a similar manner to Capecitabine via a 3 step enzymatic pathway with the final step occurring preferentially at the tumour site, formulation of the prodrug into SLNs combines the advantage of selective, localized activation with the sustained release properties of nanostructured amphiphile self-assembly and multiple payload materials thereby potentially creating a more effective anticancer agent.

Lyotropic liquid crystalline self-assembly material behavior and nanoparticulate dispersions of a phytanyl pro-drug analogue of capecitabine-a chemotherapy agent.

An amphiphile prodrug, 5'-deoxy-5-fluoro-N4-(phytanyloxycarbonyl) cytidine (5-FCPhy) has been prepared and investigated for its self-assembly material properties, in vitro cytotoxicity, and in vivo efficacy as a chemotherapy agent. The phase transitions and stability of the neat amphiphile were characterized by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). X-ray diffraction (XRD) was used to confirm the structure of the neat amphiphile, which was an amorphous glassy material. The lyotropic liquid crystalline self-assembly behavior of the amphiphile prodrug in water was examined by cross polarizing optical microscopy (POM) and small-angle X-ray scattering (SAXS). Under excess water conditions at room temperature, the amphiphile prodrug self-assembles into lyotropic liquid crystalline mesophases of inverse bicontinuous cubic symmetry. Upon aging, the inverse cubic phase slowly transformed to an inverse hexagonal phase. This amphiphile was successfully dispersed into nanoparticles of cubic and hexagonal symmetry. The in vitro cytotoxicity of dispersed nanoparticles was evaluated in seven different normal and cancer cell types and exhibited IC50 values between 70 and 90 µM for all cell types. Evaluation of 5-FCPhy in vivo against a mouse 4T1 breast tumor model displayed a trend of increasing efficacy with increasing dose. Furthermore, after 21 days, tumor volumes in the 0.5 mmol 5-FCPhy treatment group were significantly smaller than all other treatment groups including mice receiving a short chain water-soluble analogue, Capecitabine (a commercially available oral chemotherapy agent), delivered at the same dosage.

Photochemically crosslinked matrices of gelatin and fibrinogen promote rapid cell proliferation.

Here we report the use of a facile photochemical crosslinking method to fabricate stable polymer matrices from unmodified gelatin and fibrinogen. Gels were produced by covalent crosslinking of the proteins in a rapid photo-oxidative process, catalysed by a ruthenium metal complex and irradiation with visible light. For generation of macroporous, spongy matrices, the proteins and crosslinking reagents were mixed with catalase and hydrogen peroxide to achieve a foaming reaction, producing a stable, foamed matrix that was subsequently photo-crosslinked. C2C12 cells were either seeded onto the matrices after photo-curing or embedded in the protein matrix prior to foaming and crosslinking. Cells seeded onto scaffolds post-curing showed high cell viability and rapid proliferation in vitro. For cells embedded in the matrix prior to crosslinking there was some loss of initial viability, but surviving cells were able to proliferate after a period of in vitro cultivation. The matrices were shown to be biocompatible when implanted into nude mice, with evidence of proliferation and differentiation of cells seeded into the scaffolds. The results are promising for further development of tissue-engineering scaffolds based on this ruthenium-catalysed photo-crosslinking method.

A Streptococcus pyogenes derived collagen-like protein as a non-cytotoxic and non-immunogenic cross-linkable biomaterial.

A range of bacteria have been shown to contain collagen-like sequences that form triple-helical structures. Some of these proteins have been shown to form triple-helical motifs that are stable around body temperature without the inclusion of hydroxyproline or other secondary modifications to the protein sequence. This makes these collagen-like proteins particularly suitable for recombinant production as only a single gene product and no additional enzyme needs to be expressed. In the present study, we have examined the cytotoxicity and immunogenicity of the collagen-like domain from Streptococcus pyogenes Scl2 protein. These data show that the purified, recombinant collagen-like protein is not cytotoxic to fibroblasts and does not elicit an immune response in SJL/J and Arc mice. The freeze dried protein can be stabilised by glutaraldehyde cross-linking giving a material that is stable at >37 degrees C and which supports cell attachment while not causing loss of viability. These data suggest that bacterial collagen-like proteins, which can be modified to include specific functional domains, could be a useful material for medical applications and as a scaffold for tissue engineering.

Biocompatibility and modification of the protein-based adhesive secreted by the Australian frog Notaden bennetti.

When provoked, Notaden bennetti frogs secrete a proteinaceous exudate, which rapidly forms a tacky and elastic glue. This material has potential in biomedical applications. Cultured cells attached and proliferated well on glue-coated tissue culture polystyrene, but migrated somewhat slower than on uncoated surfaces. In organ culture, dissolved glue successfully adhered collagen-coated perfluoropolyether lenses to debrided bovine corneas and supported epithelial regrowth. Small pellets of glue implanted subcutaneously into mice were resorbed by surrounding tissues, and all of the animals made a full recovery. An initial but transient skin necrosis at the implant site was probably caused by some of the potentially toxic metabolites present in the frog secretion; these include sterols and carotenoids, as well as fatty alcohols, aldehydes, ketones, acids, and aromatic compounds. Removal of the carotenoid pigments did not significantly alter the glue's material properties. In contrast, peroxidase treatment of dissolved glue introduced unnatural crosslinks between molecules of the major protein (Nb-1R) and resulted in the formation of a soft hydrogel, which was very different to the original material.

Evaluation of in situ curable biodegradable polyurethanes containing zwitterion components.

Porous polyurethane networks containing covalently attached zwitterionic compounds dihydroxypolycaprolactone phosphorylcholine and 1,2-dihydroxy-N,N-dimethylamino-propane sulfonate have been prepared and characterised. Three polymers were prepared by reacting methyl 2,6-diisocyanato hexanoate functionalised D: -glucose as prepolymer A with either polycaprolactone triol alone or with addition of 10 mol% zwitterion as prepolymer B. All polymer compositions were mixed with 10 wt% hydrated gelatin beads. The cured polymers with the gelatin beads showed compression strengths that were still suitable for use in articular cartilage repair. The incorporation of zwitterions yielded more hydrophilic polymers that showed increased water absorption and increased porosity. After four months degradation in phosphate buffered saline, the polymers containing zwitterions had approximately 50% mass loss compared with 30% mass loss for that with polycaprolactone triol alone. All polymers were non-toxic in chondrocyte-based assays. Subcutaneous implantation of these polymers into rats confirmed that the polymers degraded slowly. Only a very mild inflammatory response was observed and the polymers were able to support new, well vascularised tissue formation.

Preparation of resorbable collagen-based beads for direct use in tissue engineering and cell therapy applications.

For tissue engineering and cell therapy applications, expansion of cells such as chondrocytes on beads in spinner culture can provide advantages compared with monolayer culture. The use of resorbable beads that can be included as an integral part of the construct provides the advantage of minimizing the extent of cell handling and eliminating a final trypsin treatment to detach cells from the bead. In this study, we have made various types of beads based on native collagen and denatured collagen (gelatin). The beads have been stabilized by different extents of glutaraldehyde cross-linking, and characterized by a combination of chemical analysis, thermal stability, and microscopy. In vitro examination in the presence and absence of chondrocytes showed that stability increased with the extent of crosslinking and could also be influenced by the manner of fabrication. On the basis of the in vitro stability studies, gelatin beads of a defined stability were shown to resorb over time in subcutaneous implants in nude mice compared with more stable demineralized bone particle (DMB) carriers. These data indicate that for direct use in tissue engineering or cell therapy applications, where resorbable beads can be used for cell expansion and then direct delivery of cells, it is possible to design suitable carrier beads with a range of stabilities that match the implant requirements.

A reproducible scoring system for quantification of histologic lesions of inflammatory disease in mouse gastric epithelium.

Comparison of experimental groups by microscopic examination is a common and useful method for evaluating animal models of disease. Quantification of lesions is challenging, however, and differences in scoring systems hinder comparison of results from different laboratories. The purpose of this study was to validate a simple and reproducible scoring system for Helicobacter pylori-associated gastric disease in mice. The system is based on quantification of the percentage of microscopic fields in which lesions are present, rather than on subjective estimates of lesion severity. Linear regression analyses revealed good agreement between investigators in scoring of all 3 histologic criteria examined. The range of correlation coefficients between individual readers' scores and mean scores for the 3 histologic criteria examined were: neutrophilic inflammation, 0.845 to 0.935; gastritis sufficient to displace glands, 0.919 to 0.943; and epithelial metaplasia, 0.650 to 0.799. Comparison of scores in different experimental groups by analysis of variance and Fisher least significant difference tests revealed significant differences between infected and uninfected groups and between immunodeficient and immunocompetent groups. We propose that this system may be useful in standardizing the morphologic evaluation of rodent models of H. pylori and that a similar system could be devised for evaluation of other animal models of enteric disease.

Urea, fluorofamide, and omeprazole treatments alter helicobacter colonization in the mouse gastric mucosa.

BACKGROUND: Helicobacter pylori is a causative agent of gastric and duodenal ulcers and gastric cancer. Its urease enzyme allows survival in acid conditions and drives bacterial intracellular metabolism. We aimed to investigate the role of urease in determining the intragastric distribution of Helicobacter species in vivo. MATERIALS AND METHODS: The C57BL/6 mouse model of gastritis was used for infection with Helicobacter felis (CS1) or H. pylori (SS1). Urease-modulating compounds urea and/or fluorofamide (urease inhibitor) were administered to mice over 7 days. Concurrent gastric acid inhibition by omeprazole was also examined. Bacterial distribution in the antrum, body, antrum/body, and body/cardia transitional zones was graded "blindly" by histologic evaluation. Bacterial colony counts on corresponding tissue were also conducted. RESULTS: Urease inhibition by fluorofamide decreased H. pylori survival in most gastric regions (p < .05); however, there were no marked changes to H. felis colonization after this treatment. There was a consistent trend for decreased antral colonization, and an increase in antrum/body transitional zone and body colonization with excess 5% or 6% (w/v) urea treatment. Significant reductions of both Helicobacter species were observed with the co-treatment of urea and fluorofamide (p < .05). Collateral treatment with omeprazole did not alter H. pylori colonization patterns caused by urea/fluorofamide. CONCLUSIONS: Urease perturbations affect colonization patterns of Helicobacter species. Combined urea and fluorofamide treatment reduced the density of both Helicobacter species in our infection model.

Natural colonization with Helicobacter species and the development of inflammatory bowel disease in interleukin-10-deficient mice.

BACKGROUND: The interleukin-10-deficient (IL-10-/-) mice maintained in specific-pathogen-free (SPF) conditions develop typhlocolitis when experimentally infected with Helicobacter species. However, there is limited information regarding the role of Helicobacter species that naturally colonize IL-10-/- mice in typhlocolitis development. The aim of this study was to examine in SPF IL-10-/- mice the association between natural colonization specific Helicobacter species and typhlocolitis development. MATERIAL AND METHODS: Cecum and proximal colon from 72 C57BL/6 x 129/Ola IL-10-/- mice (8-20 weeks old) were removed for DNA extraction and histologic evaluation. Genus-specific polymerase chain reaction- denaturing gradient gel electrophoresis (PCR-DGGE) and species-specific PCR were used to detect Helicobacter species. Mice were grouped by age, sex, and Helicobacter colonization status, and their histologic scores were compared. The development of clinical typhlocolitis was observed in a further 12 mice. RESULTS: Species-specific PCR showed that mice were colonized with Helicobacter ganmani and/or Helicobacter hepaticus. The PCR-DGGE detected H. ganmani, H. hepaticus and an H. ganmani-like organism. The histologic scores in mice colonized with H. hepaticus were significantly higher than that in mice colonized with H. ganmani. Male mice showed significantly higher histologic scores than female mice. Four of the 12 mice developed clinical typhlocolitis in 38 weeks. CONCLUSION: Natural colonization with different Helicobacter species was found in IL-10-/- mice within the same breeding colony. The severity of typhlocolitis differed according to the colonizing Helicobacter species. Furthermore, the rate of typhlocolitis development in IL-10-/- mice naturally colonized with Helicobacter species was significantly slower than that reported in experimentally infected mice.

Visualization of Helicobacter species within the murine cecal mucosa using specific fluorescence in situ hybridization.

BACKGROUND: Members of the genus Helicobacter have been associated with colitis development in a number of immunodeficient animal models. While it is known that these organisms can initiate colitis development, the location and spatial distribution of these bacteria within the intestinal tract is currently unknown. In this study, we developed and optimized fluorescence in situ hybridization (FISH) probes specifically for Helicobacter species. MATERIALS AND METHODS: Based on 16S-RNA gene alignments, two probes specific for the entire family Helicobacteraceae and two probes specific for Helicobacter ganmani and Helicobacter hepaticus were designed. Evaluation of these probes was determined using ATCC reference strains and cecum samples from ten IL-10 knockout mice. The presence of Helicobacter species was determined using FISH and verified using PCR-DGGE and microscopic examination of silver stained sections. RESULTS: Analysis of the ATCC reference strains revealed that the probes HEL274/HEL717 were specific for the family Helicobacteraceae, while HEP642 was specific for H. hepaticus and GAN1237 for H. ganmani. Using these probes, a pattern of spatial localization of the two different Helicobacter species was observed in the cecum tissues of IL-10 knockout mice. This consistently showed that H. ganmani was localized to the lower regions and H. hepaticus to the mid-upper regions of the crypts. CONCLUSION: We have developed FISH probes specific for the family Helicobacteraceae as well as two individual Helicobacter species. This study will allow the future use of the FISH to better understand host-pathogen interactions in vitro.

Template DNA ratio can affect detection by genus-specific PCR-denaturing gradient gel electrophoresis of bacteria present at low abundance in mixed populations.

The detection of Helicobacter species by genus-specific polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was compared with that by species-specific PCR in murine intestinal samples. Results suggest that, in samples containing multiple Helicobacter species, genus-specific PCR-DGGE may fail to detect all Helicobacter species present and that this relates to the initial template DNA ratio.

Chronic Helicobacter pylori infection with Sydney strain 1 and a newly identified mouse-adapted strain (Sydney strain 2000) in C57BL/6 and BALB/c mice.

The mouse model of Helicobacter pylori-induced disease using Sydney strain 1 (SS1) has been used extensively in Helicobacter research. Herein we describe the isolation and characterization of a new mouse-colonizing strain for use in comparative studies. One strain capable of persistent mouse colonization was isolated from a total of 110 clinical isolates and is named here SS2000 (Sydney strain 2000). Genome typing revealed a number of differences between SS1 and SS2000 as well as between them and the respective original clinical isolates. In particular, SS2000 lacked the entire cag pathogenicity island, while SS1 contained all 27 genes of the island. C57BL/6 and BALB/c mice were infected with SS1 or SS2000 or were treated with broth medium (controls). After 6 months host-specific effects were evident, including lower colonization levels in the BALB/c animals. Few pathological differences were observed between SS1- and SS2000-infected animals. However, by 15 months postinfection, SS1-infected C57BL/6 mice had developed more severe gastritis than the SS2000-infected animals. In contrast SS2000-infected BALB/c mice showed increased accumulation of mucosa-associated lymphoid tissue compared to those infected with SS1. This improved comparative model of H. pylori-induced disease allowed dissection of both host and strain effects and thus will prove useful in further studies.

Absence of mucosa-associated colonic Helicobacters in an Australian urban population.

Application of nested PCR for Helicobacter species to 416 samples obtained at colonoscopy from 15 patients with Crohn's disease, 12 with ulcerative colitis, and 43 controls revealed H. pylori DNA in only 6 individuals with no disease association. No other Helicobacter species were detected in ileal or colonic samples.

Catalase (KatA) and KatA-associated protein (KapA) are essential to persistent colonization in the Helicobacter pylori SS1 mouse model.

Helicobacter pylori infects the human gastric mucosa and elicits an aggressive inflammatory response. Despite the severity of the inflammatory response, the bacterium is able to persist and cause a chronic infection. It is believed that antioxidant defence mechanisms enable this organism to persist. Wild-type H. pylori strain SS1, and KatA- and KapA-deficient mutants, were used to infect C57/BL6 mice to test this hypothesis. Neither KatA nor KapA was essential for the initial colonization of H. pylori SS1 in the murine model of infection. The wild-type SS1 colonized the gastric mucosa at significantly higher levels than both mutants throughout the 24-week experiment. Neither KatA- nor KapA-deficient mutants were able to maintain consistent ongoing colonization for the 24-week period, indicating the necessity of both KapA and KatA in sustaining a long-term infection. At 24 weeks, 5/10 mice inoculated with the KatA mutant and 2/10 mice inoculated with the KapA mutant were colonized, compared with 10/10 of the mice inoculated with the wild-type SS1. An increase in the severity of inflammation in the wild-type-inoculated mice appeared to correlate with the decline in colonization of animals inoculated with the mutants, suggesting that increased oxidative stress militated against continued infection by the mutants. These data indicate that KapA may be of equal or greater importance than KatA in terms of sustained infection on inflamed gastric mucosae.

In vivo behavior of a Helicobacter pylori SS1 nixA mutant with reduced urease activity.

Helicobacter pylori mutants devoid of urease activity fail to colonize the gastric mucosa of mice; however, the effect of decreased levels of urease on colonization has not been examined. The nixA gene, required for full urease activity, encodes a cytoplasmic membrane nickel transporter that imports nickel ions and leads to incorporation of nickel ions into apourease. A nixA mutant of the Sydney strain of H. pylori (SS1) was constructed by disruption of the nixA gene with a kanamycin resistance cassette. This mutant retained only half the urease activity of the wild-type (wild-type) SS1 strain. C57BL/6j (n = 75) and BALB/c (n = 75) mice were inoculated independently with the wild-type or the nixA strain. The level and distribution of colonization were assessed by bacterial colony counts and histological grading at 4, 12, and 24 weeks postinfection. Colonization levels of the nixA strain in BALB/c mice were significantly lower compared with SS1 (P = 0.005), while colonization in C57BL/6j mice was similar for both the wild-type and mutant strains. Subtle differences in colonization of the different regions of the stomach, determined by microscopic grading, were observed between wild-type SS1 and the nixA strain in BALB/c mice. On the contrary, when C57BL/6j (n = 35) and BALB/c (n = 35) mice were coinfected with the wild-type and nixA strains simultaneously, the nixA mutant failed to colonize and was outcompeted by the wild-type SS1 strain, which established normal levels of colonization. These results demonstrate the importance of the nixA gene for increasing the fitness of H. pylori for gastric colonization. Since nixA is required for full urease activity, the decreased fitness of the nixA mutant is likely due to reduced urease activity; however, pleiotropic effects of the mutation cannot be completely ruled out.