[Show abstract][Hide abstract] ABSTRACT: In-vitro studies with twin-screw extruded triglyceride based implants showed promising erosion behavior. However, little is known about the behavior of such systems in-vivo and therefore a degradability study was performed in a rabbit model. Four formulations were used to prepare tsc-extrudates and implanted in rabbits and mass loss of the implants was measured after one, three and six months. Samples were additionally assessed by differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). A time dependent in-vivo erosion of the triglyceride matrices was detected and only 24% (±17%) of the matrix material was recovered after 6months of incubation (n=9). The application of a pore forming agent resulted in higher mass loss and an accelerated degradation rate. Exchange of the type of low melting triglyceride during preparation resulted in a better preservation of the porous implant structure, explained by a higher melting point of the alternate low-melting lipid. The good in-vivo degradability of tsc-extrudates was explained by the composition of the implants and the application of a low melting lipid which allowed partial melting of the implant at body temperature and rendered the implants more susceptible to degradation processes (e.g. lipolyses).
[Show abstract][Hide abstract] ABSTRACT: The influence of lipid melting on the in-vitro release of lysozyme from twin-screw extruded lipid implants was investigated. Triglyceride based implants were prepared by admixing of glycerol tristearin and various low melting lipids and subsequent twin-screw extrusion (tsc-extrusion) of these mixtures at moderate temperatures. Lysozyme was embedded as model protein and PEG 4000 or PEG 6000 was used as pore-forming excipient. By decreasing the amount of pore-forming agent from 40% to 0% lysozyme release became more sustained and the release kinetics changed from a matrix-type release profile to a linear release profile. Differential scanning calorimetry, X-ray diffraction and scanning electron microscopy measurements showed a change in implant structure upon long-term release (240days) at 37°C which was explained by partial matrix melting. In addition, partial melting of the implants was found to facilitate complete drug release at 37°C whereas at 20°C without partial melting 20% to 90% of the incorporated protein remained trapped in the implant matrix. In conclusion, partial melting of the implants during in-vitro release was found to be a major factor for the control of protein release from extruded implants and can be useful to trigger release, achieve in-vivo biodegradability and complete long-term protein release.
[Show abstract][Hide abstract] ABSTRACT: The pathways of interferon α2a release from a triglyceride based implant system were studied by single molecule fluorescence microscopy. The protein was labeled with a stable fluorescent dye ATTO647N, freeze-dried and embedded into the lipid matrix via twin-screw extrusion. The implant system consisted of a pore-forming agent (water soluble PEG 6000) and two types of triglycerides with different melting ranges which allowed the production of the implants at moderate temperatures and without the use of organic solvents. Single molecule microscopy and single particle tracking of labeled proteins contained in these implants revealed that two populations of diffusing proteins were present. Moreover, proteins were not only released via water-filled pores (created by dissolution of the pore-former), but surprisingly also through diffusion in a phase of molten lipid. Diffusion coefficients of IFNα 2a derived by tracking of individual protein molecules within the implant system were similar to diffusion coefficients obtained from control measurements in pure molten lipid and highly concentrated solutions of PEG 6000. In conclusion, tracking of individual protein molecules was successfully used to elucidate the release pathways of proteins from a relevant lipid based implant system.
[Show abstract][Hide abstract] ABSTRACT: Interferon tau (IFNT), a type I IFN similar to alpha IFNs (IFNA), is the pregnancy recognition signal produced by the ruminant conceptus. To elucidate specific effects of bovine IFNT and of other conceptus-derived factors, endometrial gene expression changes during early pregnancy were compared to gene expression changes after intrauterine application of human IFNA2. In experiment 1, endometrial tissue samples were obtained on Day (D) 12, D15, and D18 postmating from nonpregnant or pregnant heifers. In experiment 2, heifers were treated from D14 to D16 of the estrous cycle with an intrauterine device releasing IFNA2 or, as controls, placebo lipid extrudates or PBS only. Endometrial biopsies were performed after flushing the uterus. All samples from both experiments were analyzed with an Affymetrix Bovine Genome Array. Experiment 1 revealed differential gene expression between pregnant and nonpregnant endometria on D15 and D18. In experiment 2, IFNA2 treatment resulted in differential gene expression in the bovine endometrium. Comparison of the data sets from both studies identified genes that were differentially expressed in response to IFNA2 but not in response to pregnancy on D15 or D18. In addition, genes were found that were differentially expressed during pregnancy but not after IFNA2 treatment. In experiment 3, spatiotemporal alterations in expression of selected genes were determined in uteri from nonpregnant and early pregnant heifers using in situ hybridization. The overall findings of this study suggest differential effects of bovine IFNT compared to human IFNA2 and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT.
Biology of Reproduction 02/2012; 86(2):46. DOI:10.1095/biolreprod.111.094771 · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interferon-τ (IFNT), a Type-I interferon (IFN), is the pregnancy recognition signal produced by the ruminant conceptus (Godkin et al. 1984; Hansen et al. 1985; Helmer et al. 1987; Spencer et al. 2007). In addition to these specific functions of IFNT in ruminants, many studies suggest that IFNs play a general role in establishment of pregnancy and conceptus attachment/implantation in most mammalian species (Bazer et al. 2009; Bazer et al. 2010; Johnson et al. 2009; Roberts et al. 2008). To characterise the effects of prototype Type-I IFNs on bovine endometrium, in experiment one, Simmental heifers were treated from Day 14 to Day 16 of the oestrous cycle with a rod-shaped intrauterine device releasing human interferon-α (IFNA) or placebo lipid extrudates or PBS only as controls (n=4 each). Lipid formulation and concentration of human IFNA were adjusted to release 8-9×10(7) IU of IFNA over a period of 2 days in in vitro release experiments. On Day 16, endometrial biopsy samples were collected after flushing the uterus. In experiment 2, endometrial tissue samples were obtained on Day 12, 15 and 18 post-mating from nonpregnant or pregnant heifers. All samples from both experiments were analysed with an Affymetrix Bovine Genome Array (Santa Clara, CA). In experiment one, IFNA treatment resulted in differential gene expression in the bovine endometrium. Significant differences were found between the IFNA group and both control groups, whereas no differences were observed between the placebo and the PBS control group. In experiment 2, differentially expressed genes were found between pregnant and nonpregnant endometria on Day 15 and 18, but not on Day 12, with many of them known IFN-stimulated genes. The comparison of the data sets from both experiments showed very similar gene expression changes for most of the typical IFN-stimulated genes. In addition, several genes were identified which were differentially expressed after IFNA treatment but not different at Day 15 or 18 of pregnancy compared with nonpregnant animals. Conversely, some genes were found as differentially expressed during pregnancy but not after IFNA treatment. Differential expression of selected genes was verified by quantitative real-time PCR and 4 genes, namely jumonji C domain containing histone demethylase 1 homologue D (JHDM1D), indoleamine 2,3-dioxygenase 1 (IDO1), fatty acid binding protein 3, muscle and heart (mammary-derived growth inhibitor) (FABP3) and dickkopf homologue 1 (DKK1), were selected for localization of mRNA expression in endometrial tissue sections. The findings of this study suggest that there may be differential effects of bovine IFNT compared with human IFNA and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT.
Reproduction Fertility and Development 12/2011; 24(1):154. DOI:10.1071/RDv24n1Ab83 · 2.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The question whether lipid based - especially triglyceride based - depot systems can undergo biodegradation is despite many in vivo studies still unanswered. In this paper we studied biodegradation processes in vitro by incubating these lipid based systems in buffer media containing lipases. The main degradation product the free fatty acids (FFA) were isolated from the drawn samples and after derivatization analyzed with RP-HPLC. Lipid microparticles showed a rapid biodegradation whereas the complete degradation of compressed implants would take several months or years. For these two systems surface degradation can be stated. Surprisingly lipid based extrudates changed their structure dramatically upon lipase incubation resulting in a breakdown of the lipid matrix and formation of small lipid particles in the microm-range. This sort of bulk-degradation may enable the use of lipid based extrudates for the long term delivery of drugs. However additional in vivo experiments will be necessary to fully characterize these degradation processes.