Paula Weston

Effect of protein molecular weight on release from micron-sized PLGA microspheres

This study investigates the effect of protein molecular weight on release kinetics from polymeric microspheres (1-3 microm). Proteins were encapsulated at high and low loadings in poly(lactic-co-glycolic acid) (PLGA) by a phase inversion technique. Mechanism of release from this type of microsphere appeared to be dependent on protein molecular weight for microspheres with low loadings (0.5-1.6%), while independent of protein molecular weight for microspheres with high loadings (4.8-6.9%). At low loadings, release of larger proteins was dependent on diffusion through pores for the duration of the study, while smaller proteins seemed to depend on diffusion through pores initially and on degradation at later times. Following an initial diffusion phase from low loaded microspheres, lysozyme and carbonic anhydrase, the two smallest proteins, exhibited lag phases with curtailed protein release followed by a phase of increased protein release between 4 and 8 weeks, a phenomenon not evident for larger proteins. It appears that by 8 weeks, PLGA had degraded enough to allow additional release of smaller proteins which were entrapped efficiently within the microspheres. Higher loaded microspheres, which have more interconnecting channels, did not exhibit the pronounced shift from diffusion-based to polymer degradation-based release seen with the lower loaded microspheres. Interestingly, microspheres encapsulating large proteins maintained sustained release rates for 56 days.

Bioavailability, intracellular mobilization of nickel, and HIF-1α activation in human lung epithelial cells exposed to metallic nickel and nickel oxide nanoparticles

Micron-sized particles of poorly soluble nickel compounds, but not metallic nickel, are established human and rodent carcinogens. In contrast, little is known about the toxic effects of a growing number of Ni-containing materials in the nano-sized range. Here, we performed physicochemical characterization of NiO and metallic Ni nanoparticles and examined their metal ion bioavailability and toxicological properties in human lung epithelial cells. Cellular uptake of metallic Ni and NiO nanoparticles, but not metallic Ni microparticles, was associated with the release of Ni(II) ions after 24-48 h as determined by Newport Green fluorescence. Similar to soluble NiCl₂, NiO nanoparticles induced stabilization and nuclear translocation of hypoxia-inducible factor 1α (HIF-1α) transcription factor followed by upregulation of its target NRDG1 (Cap43). In contrast to no response to metallic Ni microparticles, nickel nanoparticles caused a rapid and prolonged activation of the HIF-1α pathway that was stronger than that induced by soluble Ni(II). Soluble NiCl₂ and NiO nanoparticles were equally toxic to H460 human lung epithelial cells and primary human bronchial epithelial cells; metallic Ni nanoparticles showed lower toxicity and Ni microparticles were nontoxic. Cytotoxicity induced by all forms of Ni occurred concomitant with activation of an apoptotic response, as determined by dose- and time-dependent cleavage of caspases and poly (ADP-ribose) polymerase. Our results show that metallic Ni nanoparticles, in contrast to micron-sized Ni particles, activate a toxicity pathway characteristic of carcinogenic Ni compounds. Moderate cytotoxicity and sustained activation of the HIF-1α pathway by metallic Ni nanoparticles could promote cell transformation and tumor progression.

Subfertility Caused by Altered Follicular Development and Oocyte Growth in Female Mice Lacking PKBalpha/Akt1

Abstract Mammalian females are endowed with a finite number of primordial follicles at birth. Immediately following formation of the primordial follicle pool, cohorts of follicles are either culled from the ovary or are recruited to grow until the primordial follicle population is depleted. The majority of ovarian follicles, including the oocytes, undergo atresia through apoptotic cell death. As PKBalpha/Akt1 is known to regulate apoptosis, we asked whether Akt1 functioned in the regulation of folliculogenesis in the ovary. Akt1−/− females display reduced fertility and abnormal estrous cyclicity. At Postnatal Day (PND) 25, Akt1−/− ovaries possessed a reduced number of growing antral follicles, significantly larger primary and secondary oocytes, and an increase in the number of degenerate oocytes. By PND90, there was a significant decrease in the number of primordial follicles in Akt1−/− ovaries relative to Akt1+/+. In vivo granulosa cell proliferation was reduced, as were expression levels of Kitl and Bcl2l1, two factors associated with granulosa cell proliferation/survival. No compensation was observed by Akt2 or Akt3 at the mRNA/protein level. Significantly higher serum LH and trends for lower FSH and higher inhibin A and lower inhibin B relative to Akt1+/+ females were observed in Akt1−/− females. Exposure to exogenous gonadotropins resulted in an increase in the number of secondary follicles in Akt1−/− ovaries, but few mature follicles. Collectively, our results suggest that PKBalpha/Akt1 plays an instrumental role in the regulation of the growth and maturation of the ovary, and that the loss of PKBalpha/Akt1 results in premature ovarian failure.

A 3-dimensional in vitro model of epithelioid granulomas induced by high aspect ratio nanomaterials

BackgroundThe most common causes of granulomatous inflammation are persistent pathogens and poorly-degradable irritating materials. A characteristic pathological reaction to intratracheal instillation, pharyngeal aspiration, or inhalation of carbon nanotubes is formation of epithelioid granulomas accompanied by interstitial fibrosis in the lungs. In the mesothelium, a similar response is induced by high aspect ratio nanomaterials, including asbestos fibers, following intraperitoneal injection. This asbestos-like behaviour of some engineered nanomaterials is a concern for their potential adverse health effects in the lungs and mesothelium. We hypothesize that high aspect ratio nanomaterials will induce epithelioid granulomas in nonadherent macrophages in 3D cultures.ResultsCarbon black particles (Printex 90) and crocidolite asbestos fibers were used as well-characterized reference materials and compared with three commercial samples of multiwalled carbon nanotubes (MWCNTs). Doses were identified in 2D and 3D cultures in order to minimize acute toxicity and to reflect realistic occupational exposures in humans and in previous inhalation studies in rodents. Under serum-free conditions, exposure of nonadherent primary murine bone marrow-derived macrophages to 0.5 μg/ml (0.38 μg/cm2) of crocidolite asbestos fibers or MWCNTs, but not carbon black, induced macrophage differentiation into epithelioid cells and formation of stable aggregates with the characteristic morphology of granulomas. Formation of multinucleated giant cells was also induced by asbestos fibers or MWCNTs in this 3D in vitro model. After 7-14 days, macrophages exposed to high aspect ratio nanomaterials co-expressed proinflammatory (M1) as well as profibrotic (M2) phenotypic markers.ConclusionsInduction of epithelioid granulomas appears to correlate with high aspect ratio and complex 3D structure of carbon nanotubes, not with their iron content or surface area. This model offers a time- and cost-effective platform to evaluate the potential of engineered high aspect ratio nanomaterials, including carbon nanotubes, nanofibers, nanorods and metallic nanowires, to induce granulomas following inhalation.

Nanomechanical mechanism for lipid bilayer damage induced by carbon nanotubes confined in intracellular vesicles

Significance Recent experimental studies report correlations between carbon nanotube toxicity and tube length and stiffness. Very little is known, however, about the actual behavior of these fibrous nanomaterials inside living cells following uptake, and the fundamental mechanistic link between stiffness and toxicity is unclear. Here we reveal a nanomechanical mechanism by which sufficiently long and stiff carbon nanotubes damage lysosomes, a class of membrane-enclosed organelles found inside cells that are responsible for breaking down diverse biomolecules and debris. The precise material parameters needed to activate this unique mechanical toxicity pathway are identified through coupled theoretical modeling, molecular dynamics simulations, and experimental studies, leading to a predictive pathogenicity classification diagram that distinguishes toxic from biocompatible nanomaterials based on their geometry and stiffness. Understanding the behavior of low-dimensional nanomaterials confined in intracellular vesicles has been limited by the resolution of bioimaging techniques and the complex nature of the problem. Recent studies report that long, stiff carbon nanotubes are more cytotoxic than flexible varieties, but the mechanistic link between stiffness and cytotoxicity is not understood. Here we combine analytical modeling, molecular dynamics simulations, and in vitro intracellular imaging methods to reveal 1D carbon nanotube behavior within intracellular vesicles. We show that stiff nanotubes beyond a critical length are compressed by lysosomal membranes causing persistent tip contact with the inner membrane leaflet, leading to lipid extraction, lysosomal permeabilization, release of cathepsin B (a lysosomal protease) into the cytoplasm, and cell death. The precise material parameters needed to activate this unique mechanical pathway of nanomaterials interaction with intracellular vesicles were identified through coupled modeling, simulation, and experimental studies on carbon nanomaterials with wide variation in size, shape, and stiffness, leading to a generalized classification diagram for 1D nanocarbons that distinguishes pathogenic from biocompatible varieties based on a nanomechanical buckling criterion. For a wide variety of other 1D material classes (metal, oxide, polymer), this generalized classification diagram shows a critical threshold in length/width space that represents a transition from biologically soft to stiff, and thus identifies the important subset of all 1D materials with the potential to induce lysosomal permeability by the nanomechanical mechanism under investigation.

MCF-7 Human Breast Cancer Cells Form Differentiated Microtissues in Scaffold-Free Hydrogels

Three-dimensional (3D) cultures are increasing in use because of their ability to represent in vivo human physiology when compared to monolayer two-dimensional (2D) cultures. When grown in 3D using scaffold-free agarose hydrogels, MCF-7 human breast cancer cells self-organize to form directionally-oriented microtissues that contain a luminal space, reminiscent of the in vivo structure of the mammary gland. When compared to MCF-7 cells cultured in 2D monolayer culture, MCF-7 microtissues exhibit increased mRNA expression of luminal epithelial markers keratin 8 and keratin 19 and decreased expression of basal marker keratin 14 and the mesenchymal marker vimentin. These 3D MCF-7 microtissues remain responsive to estrogens, as demonstrated by induction of known estrogen target mRNAs following exposure to 17β-estradiol. Culture of MCF-7 cells in scaffold-free conditions allows for the formation of more differentiated, estrogen-responsive structures that are a more relevant system for evaluation of estrogenic compounds than traditional 2D models.

Encapsulation of BSA using a modified W/O/O emulsion solvent removal method.

A systematic investigation of protein encapsulation in polylactic-co-glycolic-acid (PLGA) was carried out using the formation of a w/o/o emulsion followed by solvent removal. Various factors were studied, including composition of the suspension medium and the relative amounts of aqueous phase containing protein to polymer solution. High yields of microsphere fabrication were achieved by using silicon oil containing methylene chloride as a suspension medium instead of pure silicon oil, with minimal loss of polymer and protein drug (<2%). The amount of aqueous phase influenced the process and successful encapsulation was obtained if the volume ratios of aqueous phase to polymer solution were less than 5% (v/v) at a wide range of polymer concentration (2–15% g ml−1). Protein encapsulation by this w/o/o emulsion and solvent removal method has a high yield of microsphere fabrication and protein encapsulation (98%). In addition, it provides an easy way to control the release rate of protein encapsulated in microspheres by modulating their porosity in fabrication process.

Disruption of Supv3L1 damages the skin and causes sarcopenia, loss of fat, and death

Supv3L1 is a conserved and ubiquitously expressed helicase found in numerous tissues and cell types of many species. In human cells, SUPV3L1 was shown to suppress apoptotic death and sister chromatid exchange, and impair mitochondrial RNA metabolism and protein synthesis. In vitro experiments revealed binding of SUPV3L1 to BLM and WRN proteins, suggesting a role in genome maintenance processes. Disruption of the Supv3L1 gene in the mouse has been reported to be embryonic lethal at early developmental stages. We generated a conditional mouse in which the phenotypes associated with the removal of exon 14 can be tested in a variety of tissues. Disruption mediated by a Mx1 promoter-driven Cre displayed a postnatal growth delay, reduced lifespan, loss of adipose tissue and muscle mass, and severe skin abnormalities manifesting as ichthyosis, thickening of the epidermis, and atrophy of the dermis and subcutaneous tissue. Using a tamoxifen-activatable Esr1/Cre driver, Supv3L1 disruption resulted in growth retardation and aging phenotypes, including loss of adipose tissue and muscle mass, kyphosis, cachexia, and premature death. Many of the abnormalities seen in the Mx1-Cre mice, such as hyperkeratosis characterized by profound scaling of feet and tail, could also be detected in tamoxifen-inducible Cre mice. Conditional ablation of Supv3L1 in keratinocytes confirmed atrophic changes in the skin and ichthyosis-like changes. Together, these data indicate that Supv3L1 is important for the maintenance of the skin barrier. In addition, loss of Supv3L1 function leads to accelerated aging-like phenotypes.

Akt1 protects against germ cell apoptosis in the postnatal mouse testis following lactational exposure to 6-N-propylthiouracil

Exposure to 6-propyl-2-thio-uracil (PTU), a neonatal goitrogen, leads to increased testis size and sperm production in rodents. Akt1, a gene involved in cell survival and proliferation is also phosphorylated by thyroxine (T(4)). Therefore, we examined the requirement for Akt1 in germ cell survival following PTU-induced hypothyroidism. Experiments were performed using Akt1+/+, Akt1+/-, and Akt1-/- mice. PTU was administered (0.01% w/v) via the drinking water of dams from birth to PND21. At PND15, T(4) serum levels were similar in all control groups, and significantly lower in all exposed groups with a dramatic decrease in Akt1-/- mice. PTU-exposed Akt1-/- testes displayed smaller tubules, increased apoptosis, delayed lumen formation, and increased inhibin B and AMH mRNA. Relative adult testis weights were similar in all exposure groups; however, no increase in daily sperm production was observed in PTU-exposed Akt1-/- mice. In conclusion, Akt1 contributes to the effects of thyroid hormone on postnatal testis development.

Transfection of HEK Cells via DNA-loaded PLGA and P(FASA) Nanospheres

HEK cells were transfected with the GFP gene using various vectors: naked DNA, lipofectamine, and both PLGA and P(FASA) plasmid-loaded nanospheres. All methods were assessed alone and with the use of chloroquine, a lysosomal enzyme inhibitor. Transfection efficiencies were determined and compared at various times post-incubation using a fluorescence standard curve. Neither naked DNA alone nor naked DNA and chloroquine were capable of transfecting cells. No differences were evident between lipofectamine with chloroquine and lipofectamine alone which transfected cells with a constant increase in efficiency up to 2 weeks. While transfection was not feasible with polymeric nanospheres alone, the addition of chloroquine allowed DNA released from nanospheres within cells to escape endosomal degradation and transfect the cells. The increase in transfection efficiency via nanospheres over time was exponential up to 1 week, as compared to the constant rate seen for the bolus-type administration of lipofectamine, indicating that nanospheres delivered DNA to the cells by a controlled release mechanism. Additionally, the effective dose delivered to cells via nanospheres was approximately 25% that of lipofectamine, indicating that transfection via PLGA and P(FASA) nanospheres might actually be more efficient.