Helena M. Kelly

Development of collagen-hydroxyapatite scaffolds incorporating PLGA and alginate microparticles for the controlled delivery of rhBMP-2 for bone tissue engineering.

The spatiotemporally controlled delivery of the pro-osteogenic factor rhBMP-2 would overcome most of the severe secondary effects linked to the products delivering this protein for bone regeneration. With this in mind, the aim of the present work was to develop a controlled rhBMP-2 release system using collagen-hydroxyapatite (CHA) scaffolds, which had been previously optimized for bone regeneration, as delivery platforms to produce a device with enhanced capacity for bone repair. Spray-drying and emulsion techniques were used to encapsulate bioactive rhBMP-2 in alginate and PLGA microparticles, with a high encapsulation efficiency. After incorporation of these microparticles into the scaffolds, rhBMP-2 was delivered in a sustained fashion for up to 28days. When tested in vitro with osteoblasts, these eluting materials showed an enhanced pro-osteogenic effect. From these results, an optimal rhBMP-2 eluting scaffold composition was selected and implanted in critical-sized calvarial defects in a rat model, where it demonstrated an excellent healing capacity in vivo. These platforms have an immense potential in the field of tissue regeneration; by tuning the specific therapeutic molecule to the tissue of interest and by utilizing different collagen-based scaffolds, similar systems can be developed for enhancing the healing of a diverse range of tissues and organs.

Supramolecular Hydrogels with Reverse Thermal Gelation Properties from (Oligo)tyrosine Containing Block Copolymers

Novel block copolymers comprising poly(ethylene glycol) (PEG) and an oligo(tyrosine) block were synthesized in different compositions by N-carboxyanhydride (NCA) polymerization. It was shown that PEG2000-Tyr(6) undergoes thermoresponsive hydrogelation at a low concentration range of 0.25-3.0 wt % within a temperature range of 25-50 °C. Cryogenic transmission electron microscopy (Cryo-TEM) revealed a continuous network of fibers throughout the hydrogel sample, even at concentrations as low as 0.25 wt %. Circular dichroism (CD) results suggest that better packing of the β-sheet tyrosine block at increasing temperature induces the reverse thermogelation. A preliminary assessment of the potential of the hydrogel for in vitro application confirmed the hydrogel is not cytotoxic, is biodegradable, and produced a sustained release of a small-molecule drug.

Development of a thermoresponsive chitosan gel combined with human mesenchymal stem cells and desferrioxamine as a multimodal pro-angiogenic therapeutic for the treatment of critical limb ischaemia

Critical limb ischaemia (CLI) is a debilitating ischaemic disease caused by vascular occlusion. Pro-angiogenic therapeutics have the potential to produce collateral vasculature, delaying or negating the need for amputation or invasive revascularisation. Thermoresponsive hydrogels can provide an in situ depot for the sustained release of drugs and provide protection and cohesion for encapsulated cells. Human mesenchymal stem cells (hMSCs) have demonstrated strong angiogenic potential in vitro and angiogenic efficacy in vivo. Desferrioxamine (DFO), a pharmacological activator of the pro-angiogenic hypoxia inducible factor-1α pathway, has shown pro-angiogenic efficacy in vivo. This study combined hMSCs and DFO with a thermoresponsive chitosan/β-glycerophosphate (β-GP) gel, to function as an injectable, multimodal, pro-angiogenic therapeutic for the treatment of CLI. This gel underwent a thermogelation beginning at 33°C, and provided a sustained, biologically active release of DFO over the space of seven days, whilst permitting the survival, proliferation and migration of encapsulated hMSCs. hMSCs encapsulated in gel containing a 100μM concentration of DFO displayed an upregulation in VEGF expression. The combination of hMSCs and DFO within the gel resulted in a synergistic enhancement in bioactivity, as measured by increased VEGF expression in gel-exposed human umbilical vein endothelial cells. This formulation displays significant potential as an injectable pro-angiogenic therapeutic for the treatment of CLI.

Biomaterial-Enhanced Cell and Drug Delivery: Lessons Learned in the Cardiac Field and Future Perspectives.

Heart failure is a significant clinical issue. It is the cause of enormous healthcare costs worldwide and results in significant morbidity and mortality. Cardiac regenerative therapy has progressed considerably from clinical and preclinical studies delivering simple suspensions of cells, macromolecule, and small molecules to more advanced delivery methods utilizing biomaterial scaffolds as depots for localized targeted delivery to the damaged and ischemic myocardium. Here, regenerative strategies for cardiac tissue engineering with a focus on advanced delivery strategies and the use of multimodal therapeutic strategies are reviewed.

Enhanced delivery of microRNA mimics to cardiomyocytes using ultrasound responsive microbubbles reverses hypertrophy in an in-vitro model

BACKGROUND Cardiovascular diseases (CVD) account for 36% of deaths in Europe and the United States. Gene therapy can act as a therapeutic modality for the treatment of CVD. The use of microRNA mimetics may be advantageous as they regulate important processes in health and pathology. A major hurdle for using miRNA therapies relates to site specific delivery and sufficient cellular uptake of material to achieve efficacy OBJECTIVE To assess the feasibility of ultrasound responsive microbubble mediated delivery of miR mimics to cardiomyocytes. METHODS Liposome/microbubble formulations were added to HL-1 cardiomyocytes in the presence/absence of ultrasound (US). Transfection efficacy and functionality was assessed using epifluorescent microscopy, flow cytometry and qRT-PCR. DNA Quantification post-ultrasound mediated transfection of HL-1s using microbubbles was quantified. The capability of miR-133 microbubble formulations to suppress hypertrophy were measured by quantifying changes in cell size. RESULTS Ultrasound mediated microbubble formulations enhanced intracellular delivery of miR mimics in cardiomyocytes. Both complexed/encapsulated miR-microbubble formulations delivered functional miR mimics and showed no adverse effect on cardiomyocyte viability. Furthermore, ultrasound mediated microbubble transfection of miR-133 mimics reversed cardiomyocyte hypertrophy in an in-vitro model. CONCLUSIONS This novel delivery method has the potential for further development as a targeted delivery strategy for miR therapeutics to the heart.

A collagen cardiac patch incorporating alginate microparticles permits the controlled release of hepatocyte growth factor and insulin‐like growth factor‐1 to enhance cardiac stem cell migration and proliferation

Cardiac stem cells (CSCs) represent a logical cell type to exploit as a regenerative treatment option for tissue damage accrued as a result of a myocardial infarction. However, the isolation and expansion of CSCs prior to cell transplantation is time consuming, costly and invasive, and the reliability of cell expansion may also prove to be a major obstacle in the clinical application of CSC‐based transplantation therapy after a myocardial infarction. In order to overcome this, we propose the incorporation of growth factor‐eluting alginate microparticles into collagen‐based scaffolds as an implantable biomaterial to promote the recruitment and expansion of CSCs in the myocardium. In order to obtain scaffolds able to enhance the motogenic and proliferative potential of CSCs, the aim of this work was to achieve a sustained delivery of both hepatocyte growth factor and insulin‐like growth factor‐1. Both proteins were initially encapsulated in alginate microparticles by spray drying and subsequently incorporated into a collagen scaffold. Microparticles were seen to homogeneously distribute through the interconnected scaffold pore structure. The resulting scaffolds were capable of extending the release of both proteins up to 15 days, a three‐fold increase over non‐encapsulated proteins embedded in the scaffolds. In vitro assays with isolated CSCs demonstrated that the sustained release of both bioactive proteins resulted in an increased motogenic and proliferative effect. As presently practiced, the isolation and expansion of CSCs for autologous cell transplantation is slow, expensive and difficult to attain. Thus, there is a need for strategies to specifically activate in situ the intrinsic cardiac regenerative potential represented by the CSCs using combinations of growth factors obviating the need for cell transplantation. By favouring the natural regenerative capability of CSCs, it is hypothesized that the cardiac patch presented here will result in positive therapeutic outcomes in MI and heart failure patients in the future. Copyright

A stimuli responsive liposome loaded hydrogel provides flexible on-demand release of therapeutic agents

Lysolipid-based thermosensitive liposomes (LTSL) embedded in a chitosan-based thermoresponsive hydrogel matrix (denoted Lipogel) represents a novel approach for the spatiotemporal release of therapeutic agents. The entrapment of drug-loaded liposomes in an injectable hydrogel permits local liposome retention, thus providing a prolonged release in target tissues. Moreover, release can be controlled through the use of a minimally invasive external hyperthermic stimulus. Temporal control of release is particularly important for complex multi-step physiological processes, such as angiogenesis, in which different signals are required at different times in order to produce a robust vasculature. In the present work, we demonstrate the ability of Lipogel to provide a flexible, easily modifiable release platform. It is possible to tune the release kinetics of different drugs providing a passive release of one therapeutic agent loaded within the gel and activating the release of a second LTSL encapsulated agent via a hyperthermic stimulus. In addition, it was possible to modify the drug dosage within Lipogel by varying the duration of hyperthermia. This can allow for adaption of drug dosing in real time. As an in vitro proof of concept with this system, we investigated Lipogels ability to recruit stem cells and then elevate their production of vascular endothelial growth factor (VEGF) by controlling the release of a pro-angiogenic drug, desferroxamine (DFO) with an external hyperthermic stimulus. Initial cell recruitment was accomplished by the passive release of hepatocyte growth factor (HGF) from the hydrogel, inducing a migratory response in cells, followed by the delayed release of DFO from thermosensitive liposomes, resulting in a significant increase in VEGF expression. This delayed release could be controlled up to 14days. Moreover, by changing the duration of the hyperthermic pulse, a fine control over the amount of DFO released was achieved. The ability to trigger the release of therapeutic agents at a specific timepoint and control dosing level through changes in duration of hyperthermia enables sequential multi-dose profiles. STATEMENT OF SIGNIFICANCE This paper details the development of a heat responsive liposome loaded hydrogel for the controlled release of pro-angiogenic therapeutics. Lysolipid-based thermosensitive liposomes (LTSLs) embedded in a chitosan-based thermoresponsive hydrogel matrix represents a novel approach for the spatiotemporal release of therapeutic agents. This hydrogel platform demonstrates remarkable flexibility in terms of drug scheduling and sequencing, enabling the release of multiple agents and the ability to control drug dosing in a minimally invasive fashion. The possibility to tune the release kinetics of different drugs independently represents an innovative platform to utilise for a variety of treatments. This approach allows a significant degree of flexibility in achieving a desired release profile via a minimally invasive stimulus, enabling treatments to be tuned in response to changing symptoms and complications.

A Collagen Cardiac Patch Incorporating Alginate Microparticles Permits the Controlled Release of HGF and IGF-1 to Enhance Cardiac Stem Cell Migration and Proliferation.

Cardiac stem cells (CSCs) represent a logical cell type to exploit as a regenerative treatment option for tissue damage accrued as a result of a myocardial infarction. However, the isolation and expansion of CSCs prior to cell transplantation is time consuming, costly and invasive, and the reliability of cell expansion may also prove to be a major obstacle in the clinical application of CSC‐based transplantation therapy after a myocardial infarction. In order to overcome this, we propose the incorporation of growth factor‐eluting alginate microparticles into collagen‐based scaffolds as an implantable biomaterial to promote the recruitment and expansion of CSCs in the myocardium. In order to obtain scaffolds able to enhance the motogenic and proliferative potential of CSCs, the aim of this work was to achieve a sustained delivery of both hepatocyte growth factor and insulin‐like growth factor‐1. Both proteins were initially encapsulated in alginate microparticles by spray drying and subsequently incorporated into a collagen scaffold. Microparticles were seen to homogeneously distribute through the interconnected scaffold pore structure. The resulting scaffolds were capable of extending the release of both proteins up to 15 days, a three‐fold increase over non‐encapsulated proteins embedded in the scaffolds. In vitro assays with isolated CSCs demonstrated that the sustained release of both bioactive proteins resulted in an increased motogenic and proliferative effect. As presently practiced, the isolation and expansion of CSCs for autologous cell transplantation is slow, expensive and difficult to attain. Thus, there is a need for strategies to specifically activate in situ the intrinsic cardiac regenerative potential represented by the CSCs using combinations of growth factors obviating the need for cell transplantation. By favouring the natural regenerative capability of CSCs, it is hypothesized that the cardiac patch presented here will result in positive therapeutic outcomes in MI and heart failure patients in the future. Copyright

A collagen cardiac patch incorporating alginate microparticles permits the controlled release of hepatocyte growth factor and insulin-like growth factor-1 to enhance cardiac stem cell migration and proliferation: Collagen cardiac patch permits controlled release of HGF and IGF-1

Cardiac stem cells (CSCs) represent a logical cell type to exploit as a regenerative treatment option for tissue damage accrued as a result of a myocardial infarction. However, the isolation and expansion of CSCs prior to cell transplantation is time consuming, costly and invasive, and the reliability of cell expansion may also prove to be a major obstacle in the clinical application of CSC‐based transplantation therapy after a myocardial infarction. In order to overcome this, we propose the incorporation of growth factor‐eluting alginate microparticles into collagen‐based scaffolds as an implantable biomaterial to promote the recruitment and expansion of CSCs in the myocardium. In order to obtain scaffolds able to enhance the motogenic and proliferative potential of CSCs, the aim of this work was to achieve a sustained delivery of both hepatocyte growth factor and insulin‐like growth factor‐1. Both proteins were initially encapsulated in alginate microparticles by spray drying and subsequently incorporated into a collagen scaffold. Microparticles were seen to homogeneously distribute through the interconnected scaffold pore structure. The resulting scaffolds were capable of extending the release of both proteins up to 15 days, a three‐fold increase over non‐encapsulated proteins embedded in the scaffolds. In vitro assays with isolated CSCs demonstrated that the sustained release of both bioactive proteins resulted in an increased motogenic and proliferative effect. As presently practiced, the isolation and expansion of CSCs for autologous cell transplantation is slow, expensive and difficult to attain. Thus, there is a need for strategies to specifically activate in situ the intrinsic cardiac regenerative potential represented by the CSCs using combinations of growth factors obviating the need for cell transplantation. By favouring the natural regenerative capability of CSCs, it is hypothesized that the cardiac patch presented here will result in positive therapeutic outcomes in MI and heart failure patients in the future. Copyright

Advanced Material Catheter (AMCath), a minimally invasive endocardial catheter for the delivery of fast-gelling covalently cross-linked hyaluronic acid hydrogels

Injectable hydrogels that aim to mechanically stabilise the weakened left ventricle wall to restore cardiac function or to deliver stem cells in cardiac regenerative therapy have shown promising data. However, the clinical translation of hydrogel-based therapies has been limited due to difficulties injecting them through catheters. We have engineered a novel catheter, Advanced Materials Catheter (AMCath), that overcomes translational hurdles associated with delivering fast-gelling covalently cross-linked hyaluronic acid hydrogels to the myocardium. We developed an experimental technique to measure the force required to inject such hydrogels and determined the mechanical/viscoelastic properties of the resulting hydrogels. The preliminary in vivo feasibility of delivering fast-gelling hydrogels through AMCath was demonstrated by accessing the porcine left ventricle and showing that the hydrogel was retained in the myocardium post-injection (three 200 μL injections delivered, 192, 204 and 183 μL measured). However, the mechanical properties of the hydrogels were reduced by passage through AMCath (≤20.62% reduction). We have also shown AMCath can be used to deliver cardiopoietic adipose-derived stem cell-loaded hydrogels without compromising the viability (80% viability) of the cells in vitro. Therefore, we show that hydrogel/catheter compatibility issues can be overcome as we have demonstrated the minimally invasive delivery of a fast-gelling covalently cross-linked hydrogel to the beating myocardium.