Pacheco, M. O. | Search bioRxiv

bioRxiv2023

Silk Fibroin Particles as Carriers in the Development of All-Natural Hemoglobin-Based Oxygen Carriers (HBOCs)

Oxygen therapeutics have a range of applications in transfusion medicine and disease treatment. Synthetic molecules and all-natural or semi-synthetic hemoglobin-based oxygen carriers (HBOCs) have seen success as potential circulating oxygen carriers. However, many early HBOC products were removed from the market due to side effects from excess hemoglobin in the blood stream and hemoglobin entering the tissue. To overcome these issues, research has focused on increasing the molecular diameter of hemoglobin by polymerizing hemoglobin molecules or encapsulating hemoglobin in liposomal carriers, where immune responses and circulation times remain a challenge. This work looks to leverage the properties of silk fibroin, a cytocompatible and non-thrombogenic biopolymer, known to entrap protein-based cargo, to engineer a silk fibroin-hemoglobin-based oxygen carrier (sfHBOC). Herein, an all-aqueous solvent evaporation technique was used to form silk fibroin particles with and without hemoglobin to tailor the formulation for specific particle sizes. The encapsulation efficiency and ferrous state of hemoglobin were analyzed, resulting in 60% encapsulation efficiency and a maximum of 20% ferric hemoglobin, yielding 100 {micro}g/mL active hemoglobin in certain sfHBOC formulations. The system did not elicit a strong inflammation response in vitro, demonstrating the potential for this particle system to serve as an injectable HBOC. Table of Contents Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/530637v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@75421aorg.highwire.dtl.DTLVardef@46c895org.highwire.dtl.DTLVardef@11ac0ceorg.highwire.dtl.DTLVardef@1682663_HPS_FORMAT_FIGEXP M_FIG C_FIG Table of Contents Figure: In this manuscript, we generate silk fibroin particles using an all-aqueous processing technique starting from silk fibroin polymer systems of differing molecular weights. We analyze the extent to which silk concentration and extraction time affect particle size. Further, we analyze the encapsulation of hemoglobin in the particle system and assess immune activation in macrophage-like cultures.

bioengineering↗

bioRxiv2022

Impact of bioactive molecule inclusion in lyophilized silk scaffolds varies between in vivo and in vitro assessments

Biomaterials can influence the coordinated efforts required to achieve tissue rehabilitation. Sponge-like silk fibroin scaffolds that include bioactive molecules have been shown to influence tissue repair. However, the mechanisms by which scaffold formulations elicit desired in vivo responses is unclear. Here, acellular silk scaffolds consisting of type I collagen, heparin, and/or vascular endothelial growth factor (VEGF) were used to investigate material fabrication and composition parameters that drive scaffold degradation, cell infiltration, and adipose tissue deposition in vivo. In subcutaneous implants, scaffold degradation was assessed, and results show that the percentage of cells infiltrating the scaffold increased when scaffold formulations contained bioactive molecules. To gain further insight, calculated in vitro enzymatic degradation rates increased with higher enzyme concentrations and theoretical cleavage sites. However, the addition of type I collagen and heparin to the scaffold at relevant concentrations did not change degradation rates, compared to silk alone. These in vitro results are contrary to observations in vivo, where bioactive molecules influence local protein deposition, immune cell infiltration rates, and vascularization. Thus, quantitative in vitro and in vivo evaluations aid in determining the mechanisms by which biomaterials influence tissue repair and support intentional biomaterial design for clinical applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/493207v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@7b403forg.highwire.dtl.DTLVardef@1b6ed79org.highwire.dtl.DTLVardef@a0b439org.highwire.dtl.DTLVardef@9843f3_HPS_FORMAT_FIGEXP M_FIG C_FIG This work examines the role of scaffold fabrication and bioactive molecule inclusion on the enzymatic degradation of silk fibroin-based lyophilized sponges. Specifically, the roles of collagen I, heparin, and vascular endothelial growth factor are analyzed to determine the impact of formulation on rate of degradation. In addition, scaffolds are either pre-fabricated, where these bioactive molecules are included in the polymer solution prior to casting the scaffold or the bioactive molecules are introduced following scaffold formation through passive adsorption to the silk fibroin scaffold surface. Scaffolds are enzymatically degraded in vitro, and kinetic rate constants are calculated for the different formulations. In vivo, cellularity, adipose tissue accumulation, and scaffold area are assessed over time. Additionally, immunohistochemistry is used to visualize VEGF Receptor 2, CD 68, and -smooth muscle actin over time.

bioengineering↗