Francois Berthiaume - US grants

ABSTRACT Although several therapeutic options to treat chronic diabetic wounds exist, ranging from occlusive dressings, vacuum assisted closure, skin grafts, to bioengineered skin substitutes, in many instances the wounds fail to adequately respond to treatment. Chronic wounds are characterized by a failure to progress from the pro- inflammatory to the proliferative phases of wound healing. While it has been proposed to provide exogenous growth factors to the wound to help in this transition, there has been very little success using such an approach in practice. Peptide growth factors are rapidly degraded due to the overabundance of proteases in such wounds. Furthermore, recent evidence suggests that the increased levels of advanced glycation endproducts (AGEs) in the diabetic environment may interfere with signaling pathways thus making target cells poorly responsive to bioactive peptides (such as growth factors and chemokines). We have recently shown that these responses can be restored by blocking the receptor to AGEs (RAGE) using soluble RAGE (sRAGE). We propose to develop a multi-functional nanoparticle system consisting of fusion proteins of elastin-like peptides (ELPs) with relevant bioactive peptides and sRAGE. We hypothesize that these nanoparticles can exclude proteases, protecting the attached biopeptides from degradation, and that the simultaneous release of sRAGE can restore signaling in the diabetic wound. Furthermore, these nanoparticles spontaneously and reversibly self-assemble at physiological temperatures, thus enabling rapid and inexpensive purification of the fusion proteins, and with a size below 1 micrometer, nanoparticles are small enough to be easily incorporated into topical treatment modalities, including advanced methods (e.g. skin substitutes, which typically have pore sizes in excess of 50 micrometers). To test the hypothesis, we will develop a sRAGE-ELP fusion protein and combine it with one of three different bioactive peptides that target different aspects of the wound healing process: KGF-ELP (epidermis), SDF-ELP (dermis), and ARA290-ELP (tissue protective response). Our specific aims are: (1) To develop sRAGE-ELP fusion proteins that reversibly form nanoparticles with themselves and other peptide-ELP fusion proteins. (2) To evaluate the biological activity of ELP-based nanoparticles in a simulated diabetic environment in vitro. (3) To test the effect of sRAGE-ELP nanoparticles in in vivo diabetic wound conditions.

ABSTRACT Bioartificial liver assist devices (BLADs) typically consist of liver parenchymal cells (differentiated hepatocytes or hepatoma cells) cultured within an extracorporeal system (in this application, a hollow fiber (HF) bioreactor) in order to recapitulate a broad range of differentiated liver functions. Several challenges must still be overcome to realize a viable BLAD, including provision of appropriate oxygenation to the cells in the device. Our work in this area demonstrates that supplementation of hemoglobin-based O2 carriers (HBOCs) into the circulating culture medium of a HF-based BLAD represents a feasible strategy to improve O2 transport to both preserve hepatocyte differentiated functions and maintain a high cell density. However, our prior studies have shown that unmodified bovine hemoglobin (BvHb) is cytotoxic at high concentrations (>15 g/L, see preliminary data). BvHb is a tetrameric molecule (?2?2, Mw ~64 kDa) that exists in equilibrium (KD = 0.2 µM) with ?? dimers (Mw ~32 kDa) in aqueous solution. Because of the small size of the ?? dimers (32 kDa) in relation to the molecular weight cut-off (MWCO) of typical HF membranes (>35 kDa), these molecules are able to extravasate through the HF membrane and accumulate in the extracapillary space (ECS) of the HF bioreactor which contains the hepatocytes. In the ECS, ?? dimers eventually autoxidize, unfold and release free heme into solution which is cytotoxic. In light of this knowledge, we hypothesize that supplementation of circulating culture medium in a HF-based BLAD with polymerized BvHb (PolyBvHb) will provide the benefits of unmodified BvHb, namely enhance O2 transport, improve hepatocyte differentiated functions, and support a high cell density in the ECS, but without eliciting cytotoxicity. The presence of chemical cross-links within PolyBvHb will prevent cytotoxicity via two mechanisms: (a) by preventing dissociation of ?2?2 into ?? dimers and their subsequent extravasation into the ECS; (b) by preventing globin chains from unfolding and releasing free heme into solution. Three specific aims are proposed to investigate this hypothesis: Specific Aim 1: To characterize the stability of PolyBvHb in plasma and its effect on the viability, function, and proliferation of liver parenchymal cells. Specific Aim 2: To evaluate the function of a liver cell microbioreactor with zonated functions induced via controlled O2 tension. Specific Aim 3: To optimize the zonation and scale-up the HF liver bioreactor oxygenated with PolyBvHb. The proposed studies will generate proof-of-principle data that describe, both in vitro and in a clinically relevant model, the impact of using physiologically relevant oxygen tensions through the use of PolyBvHb on bioreactor performance. These studies will therefore provide the first stepping stones towards future clinical development, which would likely involve large animal (pig) and human studies.