Development of in vitro models for the investigation of stent-cell interactions

Although stenting as a medical procedure is well established, it is clear from clinical data that there is much left to learn about the long-term impact of in-stent restenosis (ISR) and how it can be prevented. Inadequacies in traditional 2D cell culture and poor in vitro and in vivo correlation contribute to this knowledge gap. In vitro cell studies of hydrophobic drugs, frequently used in drug eluting stents to combat ISR, face difficulties associated with their low aqueous solubility. Solubilization strategies that dissolve the drugs during in vitro assays result in drug concentrations that are not representative of those that will occur in vivo. This may result in non-representative changes in cell behavior, leading to further poor in vitro and in vivo correlation. Furthermore, a large share of the in vitro studies do not consider the dynamic flow of blood through a layer of cells, as occurs in the body. ISR, or re-narrowing of blood vessels, develops through a complex cascade of cellular events and is a pathological response to stent-induced vascular injury. Stent-induced vascular injury is manifested by removal of the endothelium and phenotypic changes in the underlying medial smooth muscle cells layer. This results in pathological vascular remodelling called neointimal hyperplasia (NIH). While drug-eluting stents contain anti-proliferative agents to inhibit the proliferation of smooth muscle cells (SMC), they also delay the regrowth of the intimal endothelial cells (EC) resulting in the subsequent development of late stent thrombosis. It is proposed that promoting rapid endothelial repair can minimize formation of NIH by preventing phenotypic switch in SMCs. The work presented in this thesis focuses on: i) a novel method development to investigate existing stent coatings and their impact on vascular repair, ii) evaluation of a novel bioactive stent coating candidate and its role in promoting vascular healing and iii) the establishment of a 3D cellularized tubular vascular model to bridge the gap between in vitro and in vivo studies. In the first phase of the project, a method for the preparation of stent conditioned media for in vitro evaluation of drug eluting stents was proposed and validated. In the next phase, citric acid as a novel bioactive candidate for stent coatings was investigated by studying its impact on endothelial growth and inflammation. As a last section of this project, a 3D vascular model was established and biomechanically characterized

Lipid cubic systems for sustained and controlled delivery of antihistamine drugs

Antihistamines are capable of blocking mediator responses in allergic reactions including allergic rhinitis and dermatological reactions. By incorporating various H1 receptor antagonists into a lipid cubic phase network, these active ingredients can be delivered locally over an extended period of time owing to the mucoadhesive nature of the system. Local delivery can avoid inducing unwanted side effects, often observed after systematic delivery. Lipid-based antihistamine delivery systems are shown here to exhibit prolonged release capabilities. In vitro drug dissolution studies investigated the extent and release rate of two model first generation and two model second-generation H1 antagonist antihistamine drugs from two monoacyglycerol-derived lipid models. To optimize the formulation approach, the systems were characterized macroscopically and microscopically by small-angle X-ray scattering and polarized light to ascertain the mesophase accessed upon an incorporation of antihistamines of varying solubilities and size. The impact of encapsulating the antihistamine molecules on the degree of mucoadhesivity of the lipid cubic systems was investigated using multiparametric surface plasmon resonance. With the ultimate goal of developing therapies for the treatment of allergic reactions, the ability of the formulations to inhibit mediator release utilizing RBL-2H3 mast cells with the propensity to release histamine upon induction was explored, demonstrating no interference from the lipid excipient on the effectiveness of the antihistamine molecules

Stent conditioned media for in vitro evaluation of hydrophobic stent coatings

In vitro cell studies of hydrophobic drugs face difficulties associated with their low aqueous solubility. To study poorly soluble drugs in bio-relevant media, solubilizing agents are frequently used to make stock solutions before final reconstitution in media. This results in drug concentrations that are not representative of in vivo conditions and may pose adverse effects on cells’ biological functions. This is especially true of typical hydrophobic stent coatings intended for vascular applications, where poor in vitro to in vivo correlation exists. To this end, a method for preparation of hydrophobic drug suspensions in bio-relevant media via stent conditioned media using paclitaxel (PTX) as a model drug is proposed. Since the drug is present as a suspension, this media was validated for its content uniformity and potency to induce formation of micronuclei, typical of cells undergoing prolonged mitotic arrest. Further, PTX uptake by endothelial cells was quantified and showed that the PTX stent conditioned media (at a theoretical concentration of 100 μM) suppressed cellular growth equivalent to the 0.1 μM DMSO dissolved PTX

Citric acid functionalized nitinol stent surface promotes endothelial cell healing.

While drug-eluting stents containing anti-proliferative agents inhibit proliferation of smooth muscle cells (SMCs), they also delay the regrowth of the endothelial cells which can result in subsequent development of restenosis. Acidic extracellular environments promote cell anchorage and migration by inducing conformational change in integrins, the main cell adhesion proteins. This study addresses the feasibility of a citric acid (CA) functionalized nitinol stent for improving vascular biocompatibility, specifically enhancing endothelialization. CA functionalized nitinol vascular stents are compared to commercial bare metal (Zilver Flex) and paclitaxel eluting stents (Zilver PTX) in terms of re-endothelialization. To study the effect of stent coatings, a stent conditioned media methodology was developed in an attempt to represent in vivo conditions. Overall, distinct advantages of the CA functionalized nitinol stent over commercial Zilver PTX DES and Zilver Flex BMS stents in terms of endothelial cell adhesion, migration, and proliferation are reported. These novel findings indicate the potential of a CA functionalized stent to serve as a bioactive and therapeutic surface for re-endothelialization, perhaps in combination with a SMC proliferation inhibitor coating, to prevent restenosis