Sequential Release of mRNA Complex and T Cells by a Double-Layered Implantable Scaffold for Combination Therapy of Head and Neck Squamous Cell Carcinoma.

Background: Head and neck squamous cell carcinoma (HNSCC) is associated with considerable morbidity and mortality, necessitating the development of novel therapeutic strategies. Adoptive cell therapy (ACT) and gene therapy are validated strategies for HNSCC treatment; however, both strategies have limitations when used alone in HNSCC and their synergistic integration requires further exploration to establish effective delivery platforms. In this study, we designed a double-layered scaffold based on photocurable hydrogel to combine mRNA gene therapy and ACT. And we aimed to validate the potential of this co-delivery scaffold in HNSCC treatment. Methods: An implantable, double-layered, spherical scaffold was designed using a photocurable hydrogel gelatin methacryloyl (GelMA) with suitable mechanical strength and compressive properties. This scaffold incorporated primary T cells derived from mouse lymph nodes within the inner layer and a DOTAP-mPEG-PCL (DMP)/mBim mRNA-based gene therapy complex within the outer layer. Characteristics of the scaffold and delivery capacity of DMP nanoparticles were first measured. Then the active functions of both therapeutic components were tested separately. The synergistic therapeutic efficacy of the scaffold was further validated using mouse subcutaneous and mandibular invasion models. The immune activation and killing processes associated with sequential release were measured in this process. Results: A double-layered spherical scaffold was produced and the DMP-mBim complex was characterized. The proliferation-inhibiting effect of the gene therapy complex on HNSCC cells was first demonstrated in vitro upon release, and the maintenance of T-cell bioactivity was confirmed. Results revealed the release process of two components during degradation of scaffold. The initially released DMP-mBim complex could induce immunogenic tumor cell death. Subsequently, tumor antigens generated during this process migrated into the scaffold along with the recruited dendritic cells (DCs). Activated T cells within the inner layer subsequently exerted tumor-killing effects after release. In HNSCC subcutaneous tumor and mandibular invasion models, local implantation of the double-layered scaffold effectively harnessed the synergistic effects of gene and cell therapies, inhibiting tumor growth and progression. Conclusion: The combination of DMP-mBim gene complex and T-cell therapies represents an effective immunotherapeutic strategy, and the sequential release of mRNA gene therapy and T cells within a double-layered hydrogel prolongs antitumor efficacy. This strategy presents a potential immunotherapeutic approach for HNSCC that warrants further validation to support its future clinical translation.