Growth factors are important morphogenetic proteins that instruct cell behavior and guide tissue repair and renewal. Although their therapeutic potential holds great promise in regenerative medicine applications, translation of growth factors into clinical treatments has been hindered by limitations including poor protein stability, low recombinant expression yield, and suboptimal efficacy. This review highlights current tools, technologies, and approaches to design integrated and effective growth factor-based therapies for regenerative medicine applications. The first section describes rational and combinatorial protein engineering approaches that have been utilized to improve growth factor stability, expression yield, biodistribution, and serum half-life, or alter their cell trafficking behavior or receptor binding affinity. The second section highlights elegant biomaterial-based systems, inspired by the natural extracellular matrix milieu, that have been developed for effective spatial and temporal delivery of growth factors to cell surface receptors. Although appearing distinct, these two approaches are highly complementary and involve principles of molecular design and engineering to be considered in parallel when developing optimal materials for clinical applications.
Injectable biomaterials are promising as new therapies to treat myocardial infarction (MI). One useful property of biomaterials is the ability to protect and sustain release of therapeutic payloads. In order to create a platform for optimizing the release rate of cardioprotective molecules we utilized the tunable degradation of acetalated dextran (AcDex). We created microparticles with three distinct degradation profiles and showed that the consequent protein release profiles could be modulated within the infarcted heart. This enabled us to determine how delivery rate impacted the efficacy of a model therapeutic, an engineered hepatocyte growth factor fragment (HGF-f). Our results showed that the cardioprotective efficacy of HGF-f was optimal when delivered over three days post-intramyocardial injection, yielding the largest arterioles, fewest apoptotic cardiomyocytes bordering the infarct and the smallest infarcts compared to empty particle treatment four weeks after injection. This work demonstrates the potential of using AcDex particles as a delivery platform to optimize the time frame for delivering therapeutic proteins to the heart.
In the last decade, numerous growth factors and biomaterials have been explored for the treatment of myocardial infarction (MI). While pre-clinical studies have demonstrated promising results, clinical trials have been disappointing and inconsistent, likely due to poor translatability. In the present study, we investigate a potential myocardial regenerative therapy consisting of a protein-engineered dimeric fragment of hepatocyte growth factor (HGFdf) encapsulated in a shear-thinning, self-healing, bioengineered hydrogel (SHIELD). We hypothesized that SHIELD would facilitate targeted, sustained intramyocardial delivery of HGFdf thereby attenuating myocardial injury and post-infarction remodeling. Adult male Wistar rats (n=45) underwent sham surgery or induction of MI followed by injection of phosphate buffered saline (PBS), 10 μg HGFdf alone, SHIELD alone, or SHIELD encapsulating 10 μg HGFdf. Ventricular function, infarct size, and angiogenic response were assessed 4-weeks post-infarction. Treatment with SHIELD+HGFdf significantly reduced infarct size and increased both ejection fraction and borderzone arteriole density compared to the controls. Thus, sustained delivery of HGFdf via SHIELD limits post-infarction adverse ventricular remodeling by increasing angiogenesis and reducing fibrosis. Encapsulation of HGFdf in SHIELD improves clinical translatability by enabling minimally-invasive delivery and subsequent retention and sustained delivery of this novel, potent angiogenic protein analog.
Dysregulated activity of the protease matriptase is a key contributor to aggressive tumor growth, cancer metastasis, and osteoarthritis. Methods for the detection and quantification of matriptase activity and inhibition would be useful tools. To address this need, we developed a matriptase-sensitive protein biosensor based on a dimerization-dependent red fluorescent protein (ddRFP) reporter system. In this platform, two adjoining protein domains, connected by a protease-labile linker, produce fluorescence when assembled and are nonfluorescent when the linker is cleaved by matriptase. A panel of ddRFP-based matriptase biosensor designs was created that contained different linker lengths between the protein domains. These constructs were characterized for linker-specific cleavage, matriptase activity, and matriptase selectivity; a biosensor containing a RSKLRVGGH linker (termed B4) was expressed at high yields and displayed both high catalytic efficiency and matriptase specificity. This biosensor detects matriptase inhibition by soluble and yeast cell surface expressed inhibitor domains with up to a 5-fold dynamic range and also detects matriptase activity expressed by human cancer cell lines. In addition to matriptase, we highlight a strategy that can be used to create effective biosensors for quantifying activity and inhibition of other proteases of interest.
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