Lipid nanoparticle optimization for mRNA-based head and neck cancer therapy

Introduction: Oral squamous cell carcinoma (OSCC) is the primary form of head and neck cancer, originating in the oral mucosa. According to the Global Cancer Observatory, OSCC is projected to increase by about 30% by 2030. The principal treatments for oral cancer currently include surgical tumor resection followed by postoperative radiation and chemotherapy, which greatly diminish patients’ quality of life. Additionally, the dysregulated tumor microenvironment may become increasingly aggressive, leading to metastasis, where clinical treatment often proves ineffective. Given the challenges posed by OSCC and the limitations of current treatments, exploring innovative therapeutic strategies is critical. Approximately 70% of OSCC tumors have mutated or inactivated tumor protein p53, a prevalent tumor suppressor, resulting in dysfunction associated with lower survival rates, treatment resistance, and higher relapse risks. Concurrently, ciclopirox (CPX), an antifungal recognized for its anticancer and immunomodulatory potential, presents a promising avenue for cancer therapy due to its role in repolarizing M2 macrophages. However, delivering mRNA and CPX poses significant challenges due to their instability and susceptibility to enzymatic degradation, and hydrophobic properties, respectively. Lipid nanoparticles (LNPs) offer a promising solution to these challenges. LNPs are at the forefront of non-viral nanoparticle platforms for drug delivery, showcasing their versatility and efficacy with the successful deployment of mRNA vaccines against COVID-19. Our project aims to harness this technology to create a co-delivery platform encapsulating p53 mRNA and CPX. This approach leverages the tumor-suppressive function of p53 and targets the tumor microenvironment, providing a dual-action strategy to address the complex biology of OSCC.

Materials and

Methods: Ionizable lipids with unique architectures were screened for transfection efficiency in CAL-27 cells using Firefly luciferase (FLuc) mRNA, with viability and luciferase assays identifying top formulations. These underwent further in vivo testing in a CAL-27 murine xenograft model via intratumoral (IT) injections into Nu/J mice (0.2 mL, 0.1 mg/kg). The efficacy of the top LNP, E10i-494, evaluated six hours later using an in vivo imaging system (IVIS), was benchmarked against the gold standard for mRNA delivery, C12-200. Additionally, biodistribution studies were conducted in a CAL-27 orthotopic model, using IT injections (0.02 mL, 0.1 mg/kg), confirmed the efficiency of delivery using E10i-494. This LNP, encapsulating p53 mRNA, was tested across CAL-27, OECM-1, and FaDu cell lines with dosages of 5 to 250 ng per 20,000 cells for p53-mediated cell killing efficacy at 24 and 48 hours. Concurrently, CPX was encapsulated in LNPs at molar ratios of 2%, 5%, 20%, 50% alongside FLuc mRNA to assess cell killing efficacy. The impact of CPX molar ratios on LNP characteristics, including particle size and mRNA encapsulation efficiency, was quantified using dynamic light scattering and RiboGreen assays. Lastly, CPX-LNPs reformulated with p53 mRNA were evaluated for synergistic effects on cell viability in CAL-27, OECM-1, and FaDu lines at defined time points.

Results, Conclusions, and Discussions: This study validated the co-delivery of LNPs encapsulating p53 mRNA and CPX for OSCC. Preliminary screenings showed that E10i-494 exhibited 1.5-fold higher luminescence than other LNPs in CAL-27 cultures, and a 10-fold luminescence increase over the gold standard in xenograft models, highlighting its enhanced tumor penetration and retention. Utilizing an orthotopic model of OSCC IT injections specifically transfected tumors and cervical lymph nodes, the latter is critical for OSCC's tendency to metastasize, suggesting this method could reduce off-target effects and improve patient quality of life. The distribution pattern emphasizes the LNP's potential in preventing early-stage metastasis and addressing high-risk metastatic areas. Further assessments demonstrated E10i-494's capability to effectively kill cells at low doses, with significant variations in efficacy between cell lines due to differing p53 mutation statuses. At 48 hours, a 20 ng dose led to around 50% reduction in cell viability for both CAL-27 and OECM-1, emphasizing p53 mRNA restoration's impact. At the same time point and dosage, CPX integration further enhanced this, halving viability to about 25% in both lines, underscoring the synergy between CPX and p53 mRNA. This approach exploits tumor vulnerabilities, enhancing treatment efficacy against heterogeneous OSCC. This represents the first LNP application in the oral cavity and the inaugural use of mRNA therapy for oral cancer. The delivery via IT injection could also address metastatic tumors via LNP sequestration to the lymph nodes, opening new preventative avenues. Future work will focus on monitoring in vivo tumor reduction through longitudinal studies to assess the long-term efficacy of the therapy and its impact on tumor progression. Additionally, detailed investigations into the tumor microenvironment, particularly the role of macrophage polarization into M1 macrophages, will be conducted. Understanding how LNPs influence this polarization and the overall immune response within the tumor milieu may further enhance the therapeutic potential of this dual-action strategy, aiming to improve OSCC treatment outcomes and survival rates.

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