Accelerated angiogenesis is crucial

Accelerated angiogenesis is crucial in diabetic wound healing as it provides oxygen and nutrients to the impaired tissue, alleviating uncontrolled inflammation.[1] Delivery of exogenous angiogenic growth factors (GFs), such as vascular endothelial growth factor (VEGF), is beneficial for wound closure and angiogenesis. However, direct application of VEGF has not shown clear benefits in clinical trials,[2]possibly due to the insufficient VEGF or its instability in highly proteolytic and oxidative environment of the chronic wound.[3] The most commonly used for VEGF delivery is a heparin-based hydrogel, which has a significant drawback of a burst release in the first few hours, thus requiring the addition of large amounts of growth factors in order to maintain the long-term release.[4] This may cause local or systemic side-effect profiles including aberrant angiogenesis, hemangioma, and tumorigenesis due to the excessive burst release.[5] Therefore, to overcome these limitations, we sought to develop a delivery system that can continuously produce and release VEGF, which can directly stimulate angiogenesis and subsequent regeneration of the impaired diabetic wound.Rapidly developing synthetic biology has enabled the application of living bacteria as biomolecule factories to treat metabolic diseases, infections, and cancer.[10] For example, Hay et al. demonstrated an engineered bacteria expressing fibronectin (FN) and bone morphogenetic protein-2 (BMP-2) that can control stem cell growth and differentiation.[11] Gurbatri et al. engineered a probiotic bacteria system for controlled production and intratumoral release of nanobodies targeting programmed cell death-ligand 1 (PD-L1) and cytotoxic T lymphocyte-associated protein-4 (CTLA-4), resulting in tumor egression.[12]We proposed that nonpathogenic bacteria, specifically the lactic bacteria Lactococcus lactis,[13] can be programmed with a designed gene circuit for encoding and secreting the VEGF. The lactic acid secreted by L. lactis could act as a metabolite signaling molecule to induce M1 macrophages toward M2-like polarization,[14] thereby reversing the inflammatory and proteolytic characteristic of diabetic wounds.The therapeutic effect of local administration of living bacteria is often limited by several challenges. The lack of space and environment for bacterial growth helps to reduce bacterial activity and subsequently affects production of biomolecules. Additionally, the genetically modified bacteria must be spatiotemporally restricted to reduce potential diffusion. Besides, an extracellular-mimicking environment is needed to protect and sustain release of biomolecules, especially the vulnerable growth factors. Here, a heparin-poloxamer (HP) hydrogel with some unique advantages was synthesized for the loading of L. lactis. HP is a thermosensitive polymer with lower critical solution temperature, close to human body temperature,[15]which can undergo rapid gelation with the engineered L. lactisand growth medium when applied on the wound, limiting the bacterial dispersal. The hydrogel is permeable to nutrients to support the bacterial growth and secretion of VEGF and lactic acid. Moreover, the hydrogel has a good affinity with VEGF due to the presence of heparin, which can stabilize, store, and sustain VEGF release. More important, in our microbial-based therapeutic device, the production and delivery of growth factors was simultaneous and dynamically persistent, which overcame the disadvantages of conventional heparin-functionalized delivery systems. The overall result of topical wound treatment with this on-site GF and macrophage polarization regulator codelivery system strongly promoted vascularization and accelerated wound healing (Figure 1A).