Non-small cell lung cancer (NSCLC) is a prevalent malignant tumor, accounting for over 80% of lung cancer cases, and associated with high incidence and mortality rates(Zappa and Mousa 2016). Due to the inconspicuous nature of its early symptoms and the intricate anatomical structure of the lungs, a majority of patients are diagnosed at advanced stages. Despite the availability of comprehensive treatment approaches including radiotherapy, chemotherapy, and surgery, the overall prognosis for NSCLC patients remains unsatisfactory. In recent years, extensive research has been conducted on epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) as a promising strategy to overcome the limitations of conventional therapies by specifically targeting the epidermal growth factor receptor (EGFR)(Yang et al. 2022). Represented by erlotinib and gefitinib, the first-generation EGFR-TKIs have demonstrated significant clinical efficacy in advanced NSCLC patients harboring EGFR-sensitive mutations, such as L858R and 19del, leading to an objective response rate exceeding 79% and prolonged patient survival(Li and Cui 2020). However, despite the initial success, clinical observations have revealed that the majority of patients experience disease progression within 7–13 months of first-line EGFR-TKI treatment, indicating the development of resistance to EGFR-TKIs(Yang et al. 2022; Song et al. 2023). Research has identified the T790M mutation in EGFR as a major cause of resistance to first-generation EGFR-TKIs(Zhong et al. 2023). To address the issue of acquired resistance, researchers have been continuously exploring updated iterations of EGFR-TKIs(Cooper, Sequist, Lin 2022). Nevertheless, overcoming drug resistance in lung cancer patients remains a significant challenge in clinical practice.
Afatinib (AT), an orally administered covalent second-generation EGFR-TKI, effectively inhibits the phosphorylation of EGFR and HER2 kinases in the intracellular domain, leading to downstream signaling blockade. Clinical trials have demonstrated the efficacy of combining afatinib with other antitumor agents, such as cetuximab, in the treatment of NSCLC patients who are resistant to gefitinib due to the T790M mutation(Cortot et al. 2021; Zhang et al. 2022). Cetuximab (CX) is an EGFR inhibitor that has shown efficacy in various tumor types, including head and neck tumors, colorectal cancer, and NSCLC(Brand, Iida, Wheeler 2011). It is commercially available in injectable form. Binding specifically to the extracellular domain of EGFR on tumor cells, cetuximab inhibits receptor dimerization, tyrosine kinase phosphorylation, and downstream signal transduction, transmitting apoptotic signals to the nucleus. This mechanism leads to the inhibition of cell proliferation and promotion of apoptosis(Vacchelli et al. 2014; Santos et al. 2021). However, the dose-related side effects of monoclonal antibodies often result in decreased quality of life and poor tolerability among patients. Consequently, the combination therapy of traditional oral formulations with injectable formulations has not been widely adopted in clinical practice. Nevertheless, due to cetuximab's high affinity for the extracellular domain of EGFR, it has become a prominent subject of research as a specific target for EGFR. Numerous studies have successfully incorporated cetuximab into nanocarriers to enhance the targeting capabilities of the formulation(Petrilli et al. 2018; Safaei et al. 2022).
Immunoliposomes are actively targeted liposomes that have been modified with antibodies on their surface, enabling specific recognition and enhanced targeting of tumor cells(Merino, Zalba, Garrido 2018; Singh et al. 2019). The ligands present on the surface of immunoliposomes bind to specific targets on tumor cells, forming target-ligand complexes. Upon binding stimulation, this complex is internalized via endocytosis, leading to the release of the encapsulated drug into the cytoplasm, and exerting therapeutic and cytotoxic effects. In our previous studies, we successfully conjugated cetuximab to afatinib-loaded liposomes using a thioether bond, thereby developing and evaluating a novel active targeting immunoliposomal delivery system for EGFR(Lu, Liu, Han, et al. 2019). Furthermore, injectable formulations of immunoliposomal exhibited robust drug delivery capabilities and effectively inhibited tumor growth in an NSCLC xenograft model. These findings provide evidence supporting the potential of the EGFR-targeted immunoliposomal drug delivery systems for non-small cell lung cancer treatment and pave the way for the future development of targeted formulations.
In recent years, immunoliposomes have gained increasing attention as carriers for tumor drugs, genes, vaccines, and drugs requiring penetration through the blood-brain barrier. However, several challenges still exist in the application of immunoliposomes. Firstly, the preparation process of immunoliposomes is intricate and ensuring the stability of monoclonal antibodies in novel formulations presents difficulties(Merino, Zalba, Garrido 2018). Secondly, antibody production costs are high and conventional dosages often necessitate large quantities(Bou-Assaly and Mukherji 2010; Caroline et al. 2016). Thirdly, the ability of antibody-mediated liposome internalization can impact the penetration of the drug delivery system into target cells(van Elk et al. 2016; Abu Lila and Ishida 2017). Lastly, the development of formulations is currently limited to intravenous administration, thereby restricting broader clinical applicability of immunoliposomes(Eloy et al. 2017).
Inhalable dosage forms offer distinct advantages in circumventing hepatic first-pass metabolism and enabling targeted drug delivery within the lungs, thereby minimizing systemic exposure(Shen and Minko 2020; Fei et al. 2023). Dry powder inhalation (DPI) formulations for respiratory delivery present several benefits, including enhanced stability in solid-state stability and ease of handling(Marante et al. 2020; Chang et al. 2021). Building upon our previous research, this study aims to develop a dry powder inhalation formulation of cetuximab-modified afatinib-loaded immunoliposomes for pulmonary administration. By optimizing the administration route, we aim to reduce the drug dosage while improving its therapeutic efficacy. A series of experiments has been performed to select the optimal freeze-drying process for the immunoliposomal dry powder inhalations and investigate the relevant physicochemical properties. Additionally, analysis has been performed on aerosolization performance, as well as in vitro and in vivo efficacy and safety of the novel formulation.