Development of an injectable nanomedicine-loaded hydrogel for sustained delivery of angiogenic growth factors to the ischaemic myocardium

Myocardial ischaemia, primarily occurring during and after a myocardial infarction (MI), results in cardiomyocyte death, tissue damage and eventually heart failure. A previous clinical trial identified that delivery of the angiogenic growth factor Vascular Endothelial Growth Factor (VEGF) could improve patient symptoms following MI. However, the clinical efficacy of the delivered growth factors (GFs) is hampered by their rapid in vivo clearance and degradation. Nanoparticle-based drug delivery systems that facilitate sustained growth factor delivery offer a potential solution to this problem. Traditional materials used for nanoparticle formulations, however, often require complex manufacturing techniques that result in poor encapsulation efficiency of the loaded GFs. In this thesis we propose to use bioinspired star-shaped and linear polyglutamic acid polypeptides (starPGA and linPGA) to facilitate facile encapsulation of VEGF via electrostatic interaction. The resulting nanomedicines will then be incorporated into a hydrogel suitable for minimally invasive endocardial delivery.

VEGF was successfully incorporated into both starPGA and linPGA over a range of ratios. StarPGA-VEGF 50:1 formulations produced sustained VEGF release over 28 days. StarPGA-VEGF 50:1 formulations were biocompatible and were capable of significantly improving total tubule length on a Matrigel® assay (p<0.05) and gap closure on a scratch assay (p<0.01) compared to untreated human Umbilical Vein Endothelial Cells (HUVECs). Hydrogels composed of tyramine-modified Hyaluronic Acid (HA-TA) or star-shaped poly-L-lysine (PLL) were assessed in terms of their gelation time, storage modulus, injectability and biocompatibility. On the basis of their biocompatibility and ability to remain intact over seven days in an in vitro disintegration study, four HA-TA hydrogels were chosen as lead candidates for starPGA-VEGF 50:1 incorporation.

VEGF release from HA-TA formulations containing starPGA-VEGF nanomedicines was sustained for up to 35 days, compared to just 2 days of VEGF release following incorporation of free VEGF into the same hydrogels. Repeated injections of a HA-TA + starPGA-VEGF formulation through a clinically relevant catheter were possible. The hydrogel formed following catheter injection retained its sustained release characteristic and the VEGF released from the nano-in-gel system was shown to be bioactive supporting significant improvements in total tubule length (p<0.05) and

Transwell® migration (p<0.05) compared to untreated HUVECs. Encapsulation of other GFs within the starPGA nanoparticles was achieved, in this case SDF, using the same procedure that was used for VEGF encapsulation. The chick chorioallantoic membrane (CAM) model was used to determine the ability of the developed nano-in-gel systems to promote angiogenesis in vivo. On this CAM model both HA-TA + starPGA-VEGF and HA-TA + starPGA-VEGF + starPGA-SDF formulations significantly improved vascular length density compared to both HA-TA alone and HA-TA + free VEGF. HA-TA + starPGA with no GFs loaded also significantly improved vascular length density on the CAM model compared to HA-TA alone.

Collectively the data presented in this thesis describes the formulation of a starPGA-VEGF 50:1 nanomedicine-loaded HA-TA hydrogel capable of sustained delivery of VEGF for up to 35 days in vitro. This HA-TA + starPGA-VEGF formulation can be injected through a clinically relevant catheter which would facilitate percutaneous endocardial delivery. The nano-in-gel system has shown its versatility in its ability to encapsulate and release a second angiogenic protein, SDF. In the CAM model, starPGA alone, in the absence of a loaded GF, demonstrated its ability to improve vascular length density, indicating its angiogenic potential. In this in vivo CAM model, the starPGA-VEGF nanomedicines in the HA-TA hydrogel proved significantly better than HA-TA + free VEGF at improving vascular length density.