Folate-Conjugated Quaternized Chitosan and Calcium Alginate Layer-by-Layer Nanoparticles: An Oral Platform for Targeted Gene-Immune Synergistic Therapy of Colorectal Cancer

Oral nucleic acid therapy for colorectal cancer is constrained by sequential gastrointestinal barriers and poor tumor accumulation. Herein, a barrier-aware, layer-by-layer oral nanoformulation (AMT@FH/CA NPs) is developed for co-delivery of survivin antisense oligonucleotide (ASO) and manganese ions (Mn2+) to enable combined gene-immune therapy. Core nanoparticles are formed by self-assembly of ASO, tannic acid, and Mn2+, functionalized with folic acid-conjugated quaternized chitosan for mucus penetration and tumor targeting, and then coated with a pH-responsive calcium alginate shell for gastric protection and intestinal release. AMT@FH/CA NPs are stable in gastric conditions and selectively target colonic tumor cells. The released ASO downregulates survivin expression, while Mn2+ serves as an immune adjuvant to activate the STING pathway. In an AOM/DSS colitis-associated colorectal cancer model, oral treatment reduces the tumor burden and enhances systemic immune responses without notable organ toxicity. This multi-functional nanoformulation offers a promising oral platform for nucleic acid-based combination therapy of gastrointestinal malignancies.