Nanoscale homing vectors for regenerative engineering

Abstract

Despite the improvements in treatments and outcomes following myocardial infarction (MI), cardiovascular diseases (CVDs) remain the leading cause of death worldwide. Current treatments available for patients with heart failure aim to manage pain and control symptoms but fail to regenerate the heart muscle function, resulting in reoccurrence of cardiac events and death. Stem cell therapies have been investigated for the replacement and restoration of damaged cardiac tissue, yielding improvements in myocardial function and morphology post-MI in pre-clinical studies. This was believed to occur via engraftment of stem cells at the infarct site and subsequent differentiation into new cardiomyocytes, smooth muscle cells, and endothelial cells. However, emerging evidence suggests that they do not persist at the damaged site for long, and the myocardial repair is now believed to be stimulated by paracrine effects secreted by stem cells, such as extracellular vesicles (EVs).
Consequently, recent reports have demonstrated EVs to possess both protective and regenerative capacity of the heart in animal models of MIs. However, the success of EV research is limited by a lack of standardisation, inefficient separation techniques and lack of specific markers to discriminate EV populations, low yields obtained from patient-derived samples, and unelucidated mechanism of action. Given these limitations, bioinspired artificial exosome mimics (EMs) have been explored for their use as cell-free therapeutics, allowing for a reliable and reproducible synthesis of uniform and pharmacologically suitable vesicles. Targeting of EMs to the myocardium can be achieved by modification of the lipid bilayer, and among the different techniques, the Perriman group has developed an artificial membrane binding protein (AMBP) technology, successfully applied on cells.
Here, we explore the implementation of this technology to EMs with the purpose of using them as nanoscale homing vectors for regenerative cardiac engineering. In proof-of-concept work, an AMBP previously reported by the group was successfully applied to the EM surface, characterised, and shown to bind to immobilised fibronectin in a static adhesion assay. Moreover, EMs were demonstrated to only interact with C3H mouse myoblasts (C2C12s) when modified with the AMBP, with no cytotoxicity observed. In addition, we report successful loading of the EMs with red-fluorescent protein mCherry, and subsequent delivery to cells, indicating the potential of our modified EMs to become targeted therapeutic carriers.
We then developed a modular sequential homing system in which the SpyCatcher:SpyTag partner protein system was used in conjunction with the AMBP technology. Synthesis of a ‘standard’ AMBP containing SpyCatcher would enable creation of a modular plug-and-play system in which any desired functionality could be achieved through fusion of a functional protein with the SpyTag peptide, allowing targeted homing to any desired location in vivo. Indeed, modular surface modification of the EMs was successfully executed without detriment to their structural integrity, or to the speed and efficiency of the SpyCatcher003:SpyTag003 bioorthogonal reaction. These modified EMs possessed an affinity for immobilised fibronectin and were shown to interact with C2C12s with no detriment to the cell viability, in both static experiments and under flow. Furthermore, an in vivo zebrafish model revealed accumulation of the surfaced modified EMs on the cardiac endothelium, attributed to high levels of endogenous fibronectin, with minimal off-target localisation.
Our findings highlight the potential of our new modular AMBP technology in combination with artificial exosome mimics as an off-the-shelf vehicle for therapeutic cargos that can easily be functionalised with different molecules depending on the desired outcome, as evidenced in this work using the technology as a cardiac homing vector.

Date of Award	22 Mar 2022
Original language	English
Awarding Institution	University of Bristol
Supervisor	Adam W Perriman (Supervisor) & Costanza Emanueli (Supervisor)