Balakrishnan Sivaraman

The relationship between platelet adhesion on surfaces and the structure versus the amount of adsorbed fibrinogen.

While platelet adhesion to biomaterial surfaces is widely recognized to be related to adsorbed fibrinogen (Fg), it has remained controversial whether platelet adhesion is in response to the adsorbed amount or the adsorbed conformation of this protein. To address this issue, we designed a series of platelet adhesion studies to clearly separate these two factors, thus enabling us to definitively determine whether it is the amount or the conformation of adsorbed Fg that mediates platelet response. Fg was adsorbed to a broad range of surface chemistries from a wide range of solution concentrations, with the amount and conformation of adsorbed Fg determined by absorbance and circular dichroism (CD) spectropolarimetry, respectively. Platelet adhesion response was determined by lactate dehydrogenase (LDH) assay and scanning electron microscopy (SEM). Our results show that platelet adhesion is strongly correlated with the degree of adsorption-induced unfolding of Fg (r(2)=0.96) with essentially no correlation with the amount of Fg adsorbed (r(2)=0.04). Platelet receptor inhibitor studies using an RGDS peptide reduced platelet adhesion by only about 50%, and SEM results show that adherent platelets after RGDS blocking were much more rounded with minimal extended filopodia compared with the unblocked platelets. These results provide definitive proof that the conformation of adsorbed Fg is the critical determinant of platelet adhesion, not the amount of Fg adsorbed, with adsorption-induced unfolding potentially exposing two distinctly different types of platelet binding sites in Fg; one that induces platelet adhesion alone and one that induces both platelet adhesion and activation.

Investigation of the Effects of Surface Chemistry and Solution Concentration on the Conformation of Adsorbed Proteins Using an Improved Circular Dichroism Method

In this paper we present the development of methods using circular dichroism spectropolarimetry with a custom-designed cuvette to increase the signal-to-noise ratio for the measurement of the secondary structure of adsorbed proteins, thus providing enhanced sensitivity and reproducibility. These methods were then applied to investigate how surface chemistry and solution concentration influence both the amount of adsorbed proteins and their secondary structure. Human fibrinogen and albumin were adsorbed onto alkanethiol self-assembled monolayers (SAMs) on gold with CH3, OCH2-CF3, NH2, COOH, and OH terminal groups from both dilute (0.1 mg/mL) and moderately concentrated (1.0 mg/mL) solutions. An increase in surface hydrophobicity was found to cause an increase in both the amount of the protein adsorbed and the degree of structural change that was caused by the adsorption process, while an increase in solution concentration caused an increase in the amount of protein adsorbed but a decrease in the degree of conformational change, with these effects being more pronounced on the more hydrophobic surfaces. The combined use of these two parameters (i.e., surface chemistry and solution concentration) thus provides ameans of independently varying the degree of structural change following adsorption from the amount of adsorbed protein. Further studies are underway to examine which of these factors most strongly influences platelet response, with the overall goal of developing a better understanding of the fundamental factors governing the hemocompatibility of biomaterial surfaces.

The Adherence of platelets to adsorbed albumin by receptor-mediated recognition of binding sites exposed by adsorption-induced unfolding

Although albumin (Alb) is the most abundant plasma protein, it is considered to be non-adhesive to platelets, as it lacks any known amino acid sequences for binding platelet receptors. Recent studies have suggested that platelets adhere to adsorbed Alb by mechanisms linked to its conformational state. To definitively address this issue we used circular dichroism (CD) spectropolarimetry to characterize the conformation of Alb adsorbed on a broad range of surface chemistries from a wide range of Alb solution concentrations, with platelet adhesion examined using a lactate dehydrogenase (LDH) assay and scanning electron microscopy (SEM). Our results prove that platelets bind to adsorbed Alb through receptor-mediated processes, with binding sites in Alb exposed and/or formed by adsorption-induced protein unfolding. Most importantly, beyond a critical degree of unfolding, the platelet adhesion levels correlated strongly with the adsorption-induced unfolding in Alb. The blockage of Arg-Gly-Asp (RGD) specific platelet receptors using an Arg-Gly-Asp-Ser (RGDS) peptide led to significant inhibition of platelet adhesion to adsorbed Alb, with the extent of inhibition and morphology of adherent platelets being similar for both Alb and Fg. Chemical neutralization of arginine (Arg) residues in the adsorbed Alb layer inhibited platelet-Alb interactions significantly, indicating that Arg residues play a prominent role in mediating platelet adhesion to Alb. These results provide deeper insight into the molecular mechanisms that mediate the interactions of platelets with adsorbed proteins, and how to control these interactions to improve the blood compatibility of biomaterials for cardiovascular applications.

PROBING THE CONFORMATION AND ORIENTATION OF ADSORBED ENZYMES USING SIDE-CHAIN MODIFICATION

The bioactivity of enzymes that are adsorbed on surfaces can be substantially influenced by the orientation of the enzyme on the surface and adsorption-induced changes in the enzymes structure. Circular dichroism (CD) is a powerful method for observing the secondary structure of proteins; however, it provides little information regarding the tertiary structure of a protein or its adsorbed orientation. In this study, we developed methods using side-chain-specific chemical modification of solvent-exposed tryptophan residues to complement CD spectroscopy and bioactivity assays to provide greater detail regarding whether changes in enzyme bioactivity following adsorption are due to adsorbed orientation and/or adsorption-induced changes in the overall structure. These methods were then applied to investigate how adsorption influences the bioactivity of hen egg white lysozyme (HEWL) and glucose oxidase (GOx) on alkanethiol self-assembled monolayers over a range of surface chemistries. The results from these studies indicate that surface chemistry significantly influences the bioactive state of each of these enzymes but in distinctly different ways. Changes in the bioactive state of HEWL are largely governed by its adsorbed orientation, while the bioactive state of adsorbed GOx is influenced by a combination of both adsorbed orientation and adsorption-induced changes in conformation.

Time-Dependent Conformational Changes in Adsorbed Albumin and Its Effect on Platelet Adhesion

Recent studies have shown that platelets can adhere to adsorbed albumin (Alb) through a receptor-mediated mechanism, but only if the Alb undergoes more than a critical degree of adsorption-induced unfolding. The objectives of this research were to investigate whether Alb that was initially adsorbed in a manner that induced unfolding that was less than this critical level would undergo further unfolding with time and, if so, whether this would induce the onset of platelet adhesion once this critical level was exceeded. To address these questions, CD spectropolarimetry was used to monitor the structure of Alb on OH- and CH(3)-functionalized alkanethiol self-assembled monolayer surfaces, with the Alb initially adsorbed under conditions resulting in degrees of unfolding that were below this critical level, and then the adsorbed Alb layers were aged over 6 months in sterile physiological saline at 37 °C. Platelet adhesion to Alb was quantified at selected time points via a lactate dehydrogenase (LDH) assay. The results indicate that an adsorbed Alb layer does undergo further structural changes with increasing residence time and supports platelet adhesion once it unfolds beyond the previously determined critical level. These results may be relevant to the clinically observed problem of the onset of late-thrombosis, which occurs on cardiovascular implants such as drug-eluting stents.

Multifunctional Nanoparticles for Doxycycline Delivery Towards Localized Elastic Matrix Stabilization and Regenerative Repair

Abdominal aortic aneurysms (AAAs) are abnormal expansions of the aortic wall, typically characterized by chronic up-regulation of matrix metalloproteases (MMPs)-2 and -9. These MMPs degrade elastin and elastic matrix within the aortic wall, leading to a progressive loss of elasticity of the abdominal aorta as the condition progresses. Doxycycline (DOX) is a tetracycline-based antibiotic which has shown significant promise in delaying and slowing the growth of AAAs in both clinical studies and animal models. However, it has been found to inhibit elastic matrix deposition by vascular cells at dosages in the μg ml(-1) range, which is typically observed in the circulation, in addition to systemic side effects, following oral dosage. In this paper, we describe the development of DOX-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles for localized, controlled and sustained DOX delivery towards AAA therapy. Further, we demonstrate that surface functionalization of these nanoparticles with cationic amphiphiles not only imparts them with a positive charge for potentially enhanced aortic uptake, but also enables enhanced elastin binding via hydrophobic interactions, as well as up-regulating activity of the elastin crosslinking enzyme lysyl oxidase. In addition to the DOX released from the nanoparticles being effective in inhibiting MMP-2 production and activity, we also demonstrate that surface functionalization of the nanoparticles cationic amphiphiles may also play a role in MMP-2 inhibition via (i) electrostatic interactions with negatively charged residues in the active-site of MMP-2 or (ii) steric blockade of the active site on account of the presence of two dodecyl chains in the DMAB molecule. Thus, in addition to enhanced aortic uptake and retention illustrated in studies by other groups, we have demonstrated that cationic functionalization of PLGA nanoparticles enhances elastogenic outcomes by targeted binding to elastin, as well as their potential to inhibit elastolysis. These results establish their multifunctionality as a localized delivery system for AAA therapy. Overall, this delivery system has the potential to enhance regenerative outcomes at sites of proteolytic matrix disruption/degradation by enabling targeted, controlled and long-term release of therapeutic agents.

Delineating the roles of the GPIIb/IIIa and GP-Ib-IX-V platelet receptors in mediating platelet adhesion to adsorbed fibrinogen and albumin

Platelet adhesion to adsorbed plasma proteins, such as fibrinogen (Fg), has been conventionally thought to be mediated by the GPIIb/IIIa receptor binding to Arg-Gly-Asp (RGD)-like motifs in the adsorbed protein. In previous studies, we showed that platelet adhesion response to adsorbed Fg and Alb was strongly influenced by the degree of adsorption-induced protein unfolding and that platelet adhesion was only partially blocked by soluble RGD, with RGD-blocked platelets adhering without activation. Based on these results, we hypothesized that in addition to the RGD-specific GPIIb/IIIa receptor, which mediates both adhesion and activation, a non-RGD-specific receptor set likely also plays a role in platelet adhesion (but not activation) to both Fg and albumin (Alb). To identify and elucidate the role of these receptors, in addition to GPIIb/IIIa, we also examined the GPIb-IX-V receptor complex, which has been shown to mediate platelet adhesion (but not activation) in studies by other groups. The platelet suspension was pretreated with either a GPIIb/IIIa-antagonist drug Aggrastat(®) or monoclonal antibodies 6B4 or 24G10 against GPIb-IX-V prior to adhesion on Fg- and Alb-coated OH- and CH(3)-functionalized alkanethiol self-assembled monolayer surfaces. The results revealed that GPIIb/IIIa is the primary receptor set involved in platelet adhesion to adsorbed Fg and Alb irrespective of their degree of adsorption-induced unfolding, while the GPIb-IX-V receptor complex plays an insignificant role. Overall, these studies provide novel insights into the molecular-level mechanisms mediating platelet interactions with adsorbed plasma proteins, thereby assisting the biomaterials field develop potent strategies for inhibiting platelet-protein interactions in the design of more hemocompatible cardiovascular biomaterials and effective anti-thrombotic therapies.

Nanoparticles for localized delivery of hyaluronan oligomers towards regenerative repair of elastic matrix

Abdominal aortic aneurysms (AAAs) are rupture-prone progressive dilations of the infrarenal aorta due to a loss of elastic matrix that lead to weakening of the aortic wall. Therapies to coax biomimetic regenerative repair of the elastic matrix by resident, diseased vascular cells may thus be useful to slow, arrest or regress AAA growth. Hyaluronan oligomers (HA-o) have been shown to induce elastic matrix synthesis by healthy and aneurysmal rat aortic smooth muscle cells (SMCs) in vitro but only via exogenous dosing, which potentially has side-effects and limitations to in vivo delivery towards therapy. In this paper, we describe the development of HA-o loaded poly(lactide-co-glycolide) nanoparticles (NPs) for targeted, controlled and sustained delivery of HA-o towards the elastogenic induction of aneurysmal rat aortic SMCs. These NPs were able to deliver HA-o over an extended period (>30 days) at previously determined elastogenic doses (0.2-20 μg ml(-1)). HA-o released from the NPs led to dose-dependent increases in elastic matrix synthesis, and the recruitment and activity of lysyl oxidase, the enzyme which cross-links elastin precursor molecules into mature fibers/matrix. Therefore, we were able to successfully develop a nanoparticle-based system for controlled and sustained HA-o delivery for the in vitro elastogenic induction of aneurysmal rat aortic smooth muscle cells.

Advances in biomimetic regeneration of elastic matrix structures

Elastin is a vital component of the extracellular matrix, providing soft connective tissues with the property of elastic recoil following deformation and regulating the cellular response via biomechanical transduction to maintain tissue homeostasis. The limited ability of most adult cells to synthesize elastin precursors and assemble them into mature crosslinked structures has hindered the development of functional tissue-engineered constructs that exhibit the structure and biomechanics of normal native elastic tissues in the body. In diseased tissues, the chronic overexpression of proteolytic enzymes can cause significant matrix degradation, to further limit the accumulation and quality (e.g., fiber formation) of newly deposited elastic matrix. This review provides an overview of the role and importance of elastin and elastic matrix in soft tissues, the challenges to elastic matrix generation in vitro and to regenerative elastic matrix repair in vivo, current biomolecular strategies to enhance elastin deposition and matrix assembly, and the need to concurrently inhibit proteolytic matrix disruption for improving the quantity and quality of elastogenesis. The review further presents biomaterial-based options using scaffolds and nanocarriers for spatio-temporal control over the presentation and release of these biomolecules, to enable biomimetic assembly of clinically relevant native elastic matrix-like superstructures. Finally, this review provides an overview of recent advances and prospects for the application of these strategies to regenerating tissue-type specific elastic matrix structures and superstructures.

Magnetically-responsive, multifunctional drug delivery nanoparticles for elastic matrix regenerative repair

Arresting or regressing growth of abdominal aortic aneurysms (AAAs), localized expansions of the abdominal aorta are contingent on inhibiting chronically overexpressed matrix metalloproteases (MMPs)-2 and -9 that disrupt elastic matrix within the aortic wall, concurrent with providing a stimulus to augmenting inherently poor auto-regeneration of these matrix structures. In a recent study we demonstrated that localized, controlled and sustained delivery of doxycycline (DOX; a tetracycline-based antibiotic) from poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs), enhances elastic matrix deposition and MMP-inhibition at a fraction of the therapeutically effective oral dose. The surface functionalization of these NPs with cationic amphiphiles, which enhances their arterial uptake, was also shown to have pro-matrix regenerative and anti-MMP effects independent of the DOX. Based on the hypothesis that the incorporation of superparamagnetic iron oxide NPs (SPIONs) within these PLGA NPs would enhance their targetability to the AAA site under an applied external magnetic field, we sought to evaluate the functional effects of NPs co-encapsulating DOX and SPIONs (DOX-SPION NPs) on elastic matrix regeneration and MMP synthesis/activity in vitro within aneurysmal smooth muscle cell (EaRASMC) cultures. The DOX-SPION NPs were mobile under an applied external magnetic field, while enhancing elastic matrix deposition 1.5-2-fold and significantly inhibiting MMP-2 synthesis and MMP-2 and -9 activities, compared to NP-untreated control cultures. These results illustrate that the multifunctional benefits of NPs are maintained following SPION co-incorporation. Additionally, preliminary studies carried out demonstrated enhanced targetability of SPION-loaded NPs within proteolytically-disrupted porcine carotid arteries ex vivo, under the influence of an applied external magnetic field. Thus, this dual-agent loaded NP system proffers a potential non-surgical option for treating small growing AAAs, via controlled and sustained drug release from multifunctional, targetable nanocarriers. STATEMENT OF SIGNIFICANCE Proactive screening of high risk elderly patients now enables early detection of abdominal aortic aneurysms (AAAs). There are no established drug-based therapeutic alternatives to surgery for AAAs, which is unsuitable for many elderly patients, and none which can achieve restore disrupted and lost elastic matrix in the AAA wall, which is essential to achieve growth arrest or regression. We have developed a first generation design of polymer nanoparticles (NPs) for AAA tissue localized delivery of doxycycline, a modified tetracycline drug at low micromolar doses at which it provides both pro-elastogenic and anti-proteolytic benefits that can augment elastic matrix regenerative repair. The nanocarriers themselves are also uniquely chemically functionalized on their surface to also provide them pro-elastin-regenerative & anti-matrix degradative properties. To provide an active driving force for efficient uptake of intra-lumenally infused NPs to the AAA wall, in this work, we have rendered our polymer NPs mobile in an applied magnetic field via co-incorporation of super-paramagnetic iron oxide NPs. We demonstrate that such modifications significantly improve wall uptake of the NPs with no significant changes to their physical properties and regenerative benefits. Such NPs can potentially stimulate structural repair in the AAA wall following one time infusion to delay or prevent AAA growth to rupture. The therapy can provide a non-surgical treatment option for high risk AAA patients.