Synthesis and Optimization of Collagen-targeting Peptide-Glycosaminoglycans for Inhibition of Platelets Following Endothelial Injury.

Many endothelial complications, whether from surgical or pathological origins, can result in the denudation of the endothelial layer and the exposure of collagen. Exposure of collagen results in the activation of platelets, leading to thrombotic and inflammatory cascades that ultimately result in vessel stenosis. We have previously reported the use of peptide-GAG compounds to target exposed collagen following endothelial injury. In this paper we optimize the spacer sequence of our collagen binding peptide to increase its conjugation to GAG backbones and increase the peptide-GAG collagen binding affinity by increasing peptide C-terminal cationic charge. Furthermore, we demonstrate the use of these molecules to inhibit platelet activation through collagen blocking, as well as their localization to exposed vascular collagen following systemic delivery. Altogether, optimization of peptide sequence and linkage chemistry can allow for increased conjugation and function, having implications for glycoconjugate use in other clinical applications.

Localized inhibition of platelets and platelet derived growth factor by a matrix targeted glycan mimetic significantly attenuates liver fibrosis.

New therapeutic strategies are needed for the growing unmet clinical needs in liver disease and fibrosis. Platelet activation and PDGF activity are recognized as important therapeutic targets; however, no therapeutic approach has yet addressed these two upstream drivers of liver fibrosis. We therefore designed a matrix-targeting glycan therapeutic, SBR-294, to inhibit collagen-mediated platelet activation while also inhibiting PDGF activity. Herein we describe the synthesis and characterization of SBR-294 and demonstrate its potential therapeutic benefits in vitro and in vivo. In vitro SBR-294 was found to bind collagen (EC50 = 23 nM), thereby inhibiting platelet-collagen engagement (IC50 = 60 nM). Additionally, SBR-294 was found to bind all PDGF homodimeric isoforms and to inhibit PDGF-BB mediated hepatic stellate cell activation and proliferation. Translating these mechanisms in vivo, SBR-294 reduced fibrosis by up to 54% in the CCl4 mouse model (p = 0.0004), as measured by Sirius red histological analysis. Additional fibrosis measurements were also supportive of the therapeutic benefit in this model. These results support the therapeutic benefit of platelet and PDGF antagonism and warrant further investigation of SBR-294 as a potential treatment for liver fibrosis.

Modulation of metal-azolate frameworks for the tunable release of encapsulated glycosaminoglycans.

Glycosaminoglycans (GAGs) are biomacromolecules necessary for the regulation of different biological functions. In medicine, GAGs are important commercial therapeutics widely used for the treatment of thrombosis, inflammation, osteoarthritis and wound healing. However, protocols for the encapsulation of GAGs in MOFs carriers are not yet available. Here, we successfully encapsulated GAG-based clinical drugs (heparin, hyaluronic acid, chondroitin sulfate, dermatan sulfate) and two new biotherapeutics in preclinical stage (GM-1111 and HepSYL proteoglycan) in three different pH-responsive metal-azolate frameworks (ZIF-8, ZIF-90, and MAF-7). The resultant GAG@MOF biocomposites present significant differences in terms of crystallinity, particle size, and spatial distribution of the cargo, which influences the drug-release kinetics upon applying an acidic stimulus. For a selected system, heparin@MOF, the released therapeutic retained its antithrombotic activity while the MOF shell effectively protects the drug from heparin lyase. By using different MOF shells, the present approach enables the preparation of GAG-based biocomposites with tunable properties such as encapsulation efficiency, protection and release.

Decorin mimic inhibits vascular smooth muscle proliferation and migration.

Over the past 10 years, the number of percutaneous coronary intervention procedures performed in the United States increased by 33%; however, restenosis, which inhibits complete functional recovery of the vessel wall, complicates this procedure. A wide range of anti-restenotic therapeutics have been developed, although many elicit non-specific effects that compromise vessel healing. Drawing inspiration from biologically-relevant molecules, our lab developed a mimic of the natural proteoglycan decorin, termed DS-SILY, which can mask exposed collagen and thereby effectively decrease platelet activation, thus contributing to suppression of vascular intimal hyperplasia. Here, we characterize the effects of DS-SILY on both proliferative and quiescent human SMCs to evaluate the potential impact of DS-SILY-SMC interaction on restenosis, and further characterize in vivo platelet interactions. DS-SILY decreased proliferative SMC proliferation and pro-inflammatory cytokine secretion in vitro in a concentration dependent manner as compared to untreated controls. The addition of DS-SILY to in vitro SMC cultures decreased SMC migration and protein synthesis by 95% and 37%, respectively. Furthermore, DS-SILY decreased platelet activation, as well as reduced neointimal hyperplasia by 60%, in vivo using Ossabaw swine. These results indicate that DS-SILY demonstrates multiple biological activities that may all synergistically contribute to an improved treatment paradigm for balloon angioplasty.

Collagen-binding peptidoglycans inhibit MMP mediated collagen degradation and reduce dermal scarring.

Scarring of the skin is a large unmet clinical problem that is of high patient concern and impact. Wound healing is complex and involves numerous pathways that are highly orchestrated, leaving the skin sealed, but with abnormal organization and composition of tissue components, namely collagen and proteoglycans, that are then remodeled over time. To improve healing and reduce or eliminate scarring, more rapid restoration of healthy tissue composition and organization offers a unique approach for development of new therapeutics. A synthetic collagen-binding peptidoglycan has been developed that inhibits matrix metalloproteinase-1 and 13 (MMP-1 and MMP-13) mediated collagen degradation. We investigated the synthetic peptidoglycan in a rat incisional model in which a single dose was delivered in a hyaluronic acid (HA) vehicle at the time of surgery prior to wound closure. The peptidoglycan treatment resulted in a significant reduction in scar tissue at 21 days as measured by histology and visual analysis. Improved collagen architecture of the treated wounds was demonstrated by increased tensile strength and transmission electron microscopy (TEM) analysis of collagen fibril diameters compared to untreated and HA controls. The peptidoglycan's mechanism of action includes masking existing collagen and inhibiting MMP-mediated collagen degradation while modulating collagen organization. The peptidoglycan can be synthesized at low cost with unique design control, and together with demonstrated preclinical efficacy in reducing scarring, warrants further investigation for dermal wound healing.

Incorporation of a decorin biomimetic enhances the mechanical properties of electrochemically aligned collagen threads.

Orientational anisotropy of collagen molecules is integral to the mechanical strength of collagen-rich tissues. We have previously reported a novel methodology to synthesize highly oriented electrochemically aligned collagen (ELAC) threads with mechanical properties approaching those of native tendon. Decorin, a small leucine-rich proteoglycan (SLRP), binds to fibrillar collagen and has been suggested to enhance the mechanical properties of tendon. Based on the structure of natural decorin, we have previously designed and synthesized a peptidoglycan (DS-SILY) that mimics decorin both structurally and functionally. In this study, we investigated the effect of the incorporation of DS-SILY on the mechanical properties and structural organization of ELAC threads. The results indicated that the addition of DS-SILY at a molar ratio of 30:1 (collagen:DS-SILY) significantly enhanced the ultimate stress and ultimate strain of the ELAC threads. Furthermore, differential scanning calorimetry revealed that the addition of DS-SILY at a molar ratio of 30:1 resulted in a more thermally stable collagen structure. However, addition of DS-SILY at a higher concentration (10:1 collagen:DS-SILY) yielded weaker threads with mechanical properties comparable to collagen control threads. Transmission electron microscopy revealed that the addition of DS-SILY at a higher concentration (10:1) resulted in pronounced aggregation of collagen fibrils. More importantly, these aggregates were not aligned along the long axis of the ELAC, thereby compromising the overall tensile properties of the material. We conclude that incorporation of an optimal amount of DS-SILY is a promising approach to synthesize mechanically competent collagen-based biomaterials for tendon tissue engineering applications.

The inhibition of platelet adhesion and activation on collagen during balloon angioplasty by collagen-binding peptidoglycans.

Collagen is a potent stimulator for platelet adhesion, activation, and thrombus formation, and provides a means for controlling blood loss due to injury, and recruiting inflammatory cells for fighting infection. Platelet activation is not desirable however, during balloon angioplasty/stent procedures in which balloon expansion inside an artery exposes collagen, initiating thrombosis, and inflammation. We have developed biomimetic polymers, termed peptidoglycans, composed of a dermatan sulfate backbone with covalently attached collagen-binding peptides. The peptidoglycan binds to collagen, effectively masking it from platelet activation. The lead peptidoglycan binds to collagen with high affinity (K(D) = 24 nm) and inhibits platelet binding and activation on collagen in both static studies and under flow, while promoting endothelial regrowth on collagen. Application for angioplasty is demonstrated in the Ossabaw miniature pig by fast delivery to the vessel wall through a therapeutic infusion catheter with a proprietary PTFE porous balloon. The peptidoglycan is an approach for locally preventing platelet deposition and activation on collagen. It can be used during angioplasty to prevent platelet deposition on target vessels and could be used in any vessel, including those not amenable to stent deployment.

Collagen-binding peptidoglycans: a biomimetic approach to modulate collagen fibrillogenesis for tissue engineering applications.

The small leucine-rich proteoglycans (SLRPs), prevalent in collagenous tissues, regulate collagen fibrillogenesis and provide a host of biochemical cues critical to tissue function and homeostasis. Incorporating SLRPs may enhance tissue engineering designs that mimic the native extracellular matrix, although SLRPs purified from animal sources bear low yields and lack design control. Consequently, we have designed synthetic peptidoglycans, inspired by the native SLRP decorin, that contain a collagen-binding peptide attached to a glycosaminoglycan (GAG) chain. These peptidoglycans modulate collagen fibrillogenesis and decrease fibril diameter in vitro, similarly to decorin, while maintaining the characteristic D-banded fibrils. Application for tissue engineering is demonstrated as these peptidoglycans are incorporated into collagen gels seeded with smooth muscle cells. Gels formed with peptidoglycans and decorin show a faster rate of gel compaction, and one peptidoglycan uniquely increases elastin production. The peptidoglycan design can be tailored with respect to the peptide sequence and GAG identity and is expected to have versatile application in tissue engineering.

Modification of native collagen with cell-adhesive peptide to promote RPE cell attachment on Bruch's membrane.

Current efforts to reverse loss of visual function due to Age-related Macular Degeneration point to the restoration of the Retinal Pigment Epithelial (RPE) layer. Restoration of the RPE layer involves replacing lost RPE cells as well as addressing the degeneration of the underlying Bruch's membrane (BM). To advance the potential of using donor BM, we present a strategy to achieve specific and controllable modification of the inner collagenous layer (ICL) of the Bruch's membrane. In particular, interaction between a collagen binding peptide (CBP) sequence with exposed collagen fibers on the ICL surface is utilized to anchor bioactive molecules. Here, a cell-adhesion sequence is added to the collagen binding sequence to promote attachment and survival of ARPE-19. First, the binding specificity of the CBP sequence is verified with a fluorescent binding assay. Subsequently, the effect of modification using the peptide is studied qualitatively using confocal fluorescent imaging and quantitatively through a cell proliferation assay. Results of these experiments indicate that the peptide sequence binds specifically to collagen fibers. Additionally, modification using the peptide enhanced cell adhesion, allowing large uniform cell networks to be formed on the surface. Furthermore, modification with the peptide also delayed the onset of apoptosis on adherent cells.

Design of a synthetic collagen-binding peptidoglycan that modulates collagen fibrillogenesis.

The ubiquity of collagen in mammalian tissues, with its host of structural and chemical functions, has motivated its research in many fields, including tissue engineering. The organization of collagen is known to affect cell behavior and the resulting structural integrity of tissues or tissue engineered scaffolds. Of particular interest are proteoglycan (PG) interactions with collagen and their influence on collagen assembly. These natural molecules provide unique chemical and mechanical cues and are known to modulate collagen fibrillogenesis. Research has been limited to PGs extracted and purified from animal sources and has the drawbacks of limited design control and costly purification. Consequently, we have designed a synthetic peptidoglycan based on decorin, a collagen-binding PG. The synthetic peptidoglycan containing a collagen-binding peptide with a single dermatan sulfate side chain specifically binds to collagen, delays fibrillogenesis, and increases collagen gel stiffness as decorin does. This design can be tailored with respect to the peptide sequence and attached glycosaminoglycan chain, offering unique control with relative ease of manufacturing.