# Alginate formulations with high loads of zebularine and retinoic acid promote tissue growth and innervation and induce extensive epigenetic repatterning

## TL;DR

A new therapy using zebularine and retinoic acid in alginate promotes tissue regeneration and nerve growth in mice without harmful effects.

## Contribution

A novel regenerative therapy combining zebularine and retinoic acid in an alginate formulation is developed and tested.

## Key findings

- The formulation promotes tissue architecture restoration and nerve and vessel network growth in mouse wound models.
- The therapy induces extensive epigenetic changes, including methylation of neurodevelopmental genes.
- The alginate formulation is safe, with no cytotoxicity observed in cell culture tests.

## Abstract

The study presents the development of a small-molecule epigenetic regenerative therapy that combines a demethylating agent, zebularine, with retinoic acid, acting as a transcriptional activator, and an alginate carrier. Subcutaneously injected formulations based on 2% sodium alginate containing high loads of zebularine (240 mg/ml) and retinoic acid (0.8 mg/ml) promoted regenerative responses in a mouse model of ear pinna punch wound involving the restoration of tissue architecture, the growth of nerve and vessel networks, and extensive alterations in gene methylation and expression profiles with no adverse effects in the animals. Among the remarkable changes in global gene methylation are those in neurodevelopmental genes. In vitro studies showed rapid discharge of zebularine but not retinoic acid from the alginate formulations. Live ultrasound imaging demonstrated gradual absorption of the subcutaneously injected alginate formulations, which may explain the in vivo activity of retinoic acid following subcutaneous administration. Cell culture tests exhibited no significant cytotoxicity of the alginate formulations. The simplicity of composition, preparation, and administration of alginate-based drug formulations is a distinctive advantage. The effective induction of regenerative response, together with a high safety profile of subcutaneously administered pro-regenerative alginate formulations, opens the way to testing further regenerative therapies for hard-to-reach lesions.

The online version contains supplementary material available at 10.1038/s41598-025-22528-8.