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A reduction in blood supply to any limb causes ischaemia, pain and morbidity. Critical limb ischaemia is the most serious presentation of peripheral vascular disease. One in five patients with critical limb ischaemia will die within six months of diagnosis and one in three will require amputation in this time. Improving blood flow to the limb, via the administration of angiogenic agents, could relieve pain and avoid amputation. Herein, chitosan is combined with β-glycerophosphate to form a thermoresponsive formulation (chitosan/β-GP) that will flow through a syringe and needle at room temperature but will form a gel at body temperature. The chitosan/β-GP hydrogel, with or without the angiogenic molecule desferrioxamine (DFO), was injected into the mouse hind limb, following vessel ligation, to test the ability of the formulations to induce angiogenesis. The effects of the formulations were measured using laser Doppler imaging to determine limb perfusion and CD31 staining to quantify the number of blood vessels. Twenty-eight days following induction of ischaemia, the chitosan/β-GP and chitosan/β-GP + 100 µM DFO formulations had significantly ( p < 0.001 and p < 0.05, respectively) improved blood flow in the ischaemic limb compared with an untreated control. Chitosan/β-GP increased vessel number by 1.7-fold in the thigh of the ischaemic limb compared with an untreated control, while chitosan/β-GP + 100 µM DFO increased vessel number 1.8-fold. Chitosan/β-GP represents a potential minimally invasive treatment for critical limb ischaemia.

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., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Cell delivery to the infarcted heart has emerged as a promising therapy, but is limited by very low acute retention and engraftment of cells. The objective of this study was to compare a panel of biomaterials to evaluate if acute retention can be improved with a biomaterial carrier. Cells were quantified post-implantation in a rat myocardial infarct model in five groups (n = 7-8); saline injection (current clinical standard), two injectable hydrogels (alginate, chitosan/β-glycerophosphate (chitosan/ß-GP)) and two epicardial patches (alginate, collagen). Human mesenchymal stem cells (hMSCs) were delivered to the infarct border zone with each biomaterial. At 24 h, retained cells were quantified by fluorescence. All biomaterials produced superior fluorescence to saline control, with approximately 8- and 14-fold increases with alginate and chitosan/β-GP injectables, and 47 and 59-fold increases achieved with collagen and alginate patches, respectively. Immunohistochemical analysis qualitatively confirmed these findings. All four biomaterials retained 50-60% of cells that were present immediately following transplantation, compared to 10% for the saline control. In conclusion, all four biomaterials were demonstrated to more efficiently deliver and retain cells when compared to a saline control. Biomaterial-based delivery approaches show promise for future development of efficient in vivo delivery techniques., (Copyright © 2014 Elsevier Ltd. All rights reserved.)