Melittin-Clonidine peritumoral injection gel for cold tumor: exposing tumor antigens and reducing immunosuppressive pressure on immune cells.

Immunotherapy has revolutionized the treatment of cancer by activated the immune system to achieve durable responses. Despite these successes, its efficacy remains largely constrained in immunologically "cold" tumors, characterized by poor T-cell infiltration and low mutational burden, such as triple-negative breast cancer (TNBC) and microsatellite stable colorectal cancer. To overcome this limitation, we investigated a combination strategy utilizing melittin (MEL) and clonidine (CLD). Among them, MEL belongs to a cationic amphipathic peptide, not only exhibits direct antitumor activity but also promotes the release of tumor-associated antigens, potentially initiating an immune response. Besides, CLD, an FDA-approved α2-adrenergic receptor agonist, has been shown to enhance the recruitment of CD8 T cells. Here, we developed an injectable thermosensitive hydrogel based on poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA, PPP) for spatiotemporally controlled co-delivery of MEL and CLD to tumor sites. The PPP hydrogel exhibited excellent sol-gel transition behavior at physiological temperatures, enabling localized retention and sustained release. MEL@PPP promoted apoptosis andinduced immunogenic cell death invitro. In the orthotopic 4T1 breast cancer models, the MEL/CLD-loaded hydrogel (MEL + CLD)@PPP significantly inhibited tumor growth and reshaped the immune microenvironment, such as promoting the maturation of dendritic cells and activation of T cells in tumors, as evidenced by enhanced expression of cytotoxic markers (IFN-γ and Granzyme B). Moreover, (MEL + CLD)@PPP didn't cause significant changes in blood pressure. Furthermore, when combined with an anti-PD-L1 antibody (αPD-L1), (MEL + CLD)@PPP significantly enhanced therapeutic efficacy in both 4T1 and CT26 tumor cells, alleviated T cell exhaustion and M2-type macrophage polarization. These findings suggested that the peritumoral injection of thermosensitive hydrogel showcases a "kill two birds with one stone" approach that achieves continuous release of drugs at the tumor site and boosts tumor immunogenicity, offering a novel and streamlined strategy for "cold cancer" treatment.