Pancreatic islet transplantation, which includes replacing the damaged or destroyed islets in the recipient's pancreas with healthy islets from a donor, is one of the therapeutic applications to treat type 1 diabetes mellitus (T1D). The challenges that this approach encounters, which include the fact that transplanted islets frequently do not receive enough oxygen, leading to graft failure, hypoxia-related grafts and restrict its efficacy. We developed Respiratoid, a novel oxygen-generating biomaterial, to overcome these issues by providing oxygen to transplanted islets or tissues after they have been placed in the body of a recipient and preventing immunological rejection. Respiratoid is composed of chloroplasts, organelles involved in photosynthesis that can generate oxygen; chloroplast-transit-peptide (CTP), which can be inserted into the chloroplast via the outer membrane protein transmembrane region and upregulate pathway proteins involved in oxygen generation; and alginate, a biocompatible polymer that forms hydrogels.

Using a carbodiimide coupling procedure with EDC/NHS, Alginate and CTP are conjugated via an amide bond to form Alginate-CTP conjugation polymer (Al-CTP). The Al-CTP conjugation polymer was verified by 1H NMR spectroscopy and FT-IR. To create Respiratoid hydrogels, Al-CTP is mixed in a 1:1 volume ratio with chloroplasts isolated from Spinacia oleracea, and pancreatic islets isolated from rats are encapsulated using an encapsulator and droplet cross-linking with calcium chloride solution. We confirmed that Repiratoid hydrogels generated oxygen in hypoxic conditions (1% O2) and secreted enough insulin without compromising islet viability, as assessed by glucose-stimulated insulin secretion (GSIS), CCK-8 assay, and immunostaining. Respiratoid hydrogels generated more oxygen, had higher cell viability, and secreted more insulin compared to the Alginate with chloroplasts without CTP. Importantly, we demonstrated the activity of CTP using RNA-Seq analysis, which proved that CTP upregulated gene related to molecular functions and cell components of chloroplasts related to quinone binding and electron transport chain, ultimately leading to the formation of oxygen-evolving complexes in Photosystem II, and Repiratoid biomaterial can cause hyperoxygenation. Moreover, we confirmed that subcutaneously implanted Respiratoid hydrogels generated much more oxygen, as measured using an oxygen probe sensor, and that intraperitoneally implanted allogeneic islets encapsulated in Respiratoid hydrogels were functional and could reverse diabetes in mouse for 100 days, without showing any signs of toxicity.

In summary, this novel biomaterial has the capacity to generate oxygen. Respiratoid hydrogels represent a novel tool for effective islet transplantation and the long-term treatment of T1D. Overall, our research shows that Respiratoid hydrogels have the potential to provide a platform for islet transplantation, enhance clinical results.