The work described in this dissertation presents the synthesis and characterization of a novel multivalent conjugate of mechano-growth factor. Mechano-growth factor is a peptide derived from a splice variant of insulin-like growth factor-1 protein, which has shown promising cardioprotective effects. Multivalent conjugate technology provides a platform in which growth factors of interest are multivalently conjugated to a long, soluble polymer chain. This multivalent conjugation can result in improved pharmacokinetics and decreased degradation of the growth factor, as well as potentially increasing the bioactivity of the growth factor compared to its unconjugated form. Since the benefits of multivalent conjugate technology depend heavily upon the final valency of the conjugated growth factor, as well as the size and distribution of the multivalent conjugate molecules, it was necessary to have a characterization technique that could provide this information. This work focused on using multi-angle light scattering to thoroughly characterize the mechano-growth factor conjugates, as well as other conjugate molecules and macromolecules of interest in the field of tissue engineering. Additionally, this work focused on the development of in vitro cell-based assays for use in studying the bioactivity of the mechanogrowth factor conjugates.
Chapter 3 presents the development of a reaction method to allow for the multivalent conjugation of MGF peptide to a HyA backbone chain. The conjugation reaction required first the synthesis of a HyA intermediate by the addition of acrylate groups, which were characterized through gelation and NMR spectroscopy. Then the conjugation was achieved by a Michael addition reaction between the acrylate groups on the HyA and the c-terminal cysteine on the MGF peptide. The success of this conjugation reaction was verified through a BCA assay, which also provided an estimate of the final conjugation ratios. The characterization using NMR spectroscopy, acrylated HyA gelation, and BCA was able to confirm the success of conjugation and provide estimates of the peptide concentration and final conjugation ratio.
Chapter 4 provides a more thorough characterization of the conjugate molecules through the application of multi-angle light scattering. This analysis utilized a SEC-MALS-UV-RI method, where the inline use of two concentration detectors allowed for the determination of the relative compositions of the MVCs. Using the measured specific refractive index increment and UV extinction coefficient values measured for the two MVC components, it was possible to determine the weight fractions of the MGF and HyA in the total MVC molecule. This analysis confirmed that the bioconjugate chemistry technique utilized in this work was successful, and that it was possible to determine the total molecular weight, polydispersity, conjugation efficiency, and valency of the MVCs.
Chapter 5 presents an alternative SEC-MALS methodology for characterizing MVC molecules using branching analysis. It was first demonstrated that the MVC molecules behave as branched molecules, and then it was shown that branching analysis methods could be successfully applied to the MVCs. Although a linear hyaluronic acid was used as the linear counterpart for the calculations instead of a chemically identical linear counterpart, it was still possible to achieve good agreement between the values calculated by branching analysis and multivalent conjugate analysis.
Chapter 6 further demonstrates the power of SEC-MALS as a characterization technique for macromolecules and MVCs in dilute solutions. SEC-MALS was applied to a variety of different projects in order to provide detailed information about the molecular weight, radius of gyration, polydispersity, and valency of these different macromolecular systems.
Chapter 7 focuses on the development of two different assays for assessing the bioactivity of mvMGF. The first assay used hypoxia to mimic the ischemic environment of the cardiac tissue during an MI, while the second assay stressed CMs through cryopreservation and subsequent thawing. Both assays resulted in functional impairment of the CMs, which will allow for the in vitro assessment of the cardioprotective ability of the mvMGF.