Drug Combination Nanoparticles Carrying Gemcitabine and Paclitaxel: Formation Mechanism Influencing Pharmacokinetics, Drug Metabolism, and Efficacy in Triple-Negative Breast Cancer Mouse Models

Abstract

Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer and remains a major clinical challenge due to the absence of hormone receptors and human epidermal growth factor receptor 2 (HER2), rapid disease progression, and limited treatment options. Conventional combination chemotherapeutics are effective but limited by poor drug solubility, rapid systemic clearance, and a lack of synchronized delivery. Addressing these challenges requires an advanced drug delivery system capable of synchronizing the delivery of chemotherapeutics with diverse physicochemical properties to tumors in a long-acting and in vivo stable manner. To address this need, our research team developed a drug combination nanoparticle (DcNP) that enable the co-assembly of physicochemical diverse drugs, such as hydrophilic gemcitabine (G) and hydrophobic paclitaxel (T), into a nanoparticle, referred to as GT-in-DcNP. We previously demonstrated that one GT-in-DcNP composition achieves long-acting pharmacokinetics and synchronized delivery of both drugs in mouse models. However, the impact of the formation mechanism on in vivo pharmacokinetics (PK), metabolic protection of gemcitabine, and therapeutic efficacy across different GT-in-DcNP compositions remains unclear. To address these questions, various GT-in-DcNP compositions were designed and first characterized in vitro, where they exhibited comparable physicochemical properties. These formulations were subsequently evaluated for in vivo pharmacokinetics and therapeutic efficacy in both early- and late-stage TNBC mouse models. Mechanistic studies revealed that key preparation processes, such as controlled solvent removal, are essential for enabling short-acting gemcitabine to transition into a long-acting form in vivo. Using subcutaneous administration, a selected GT-in-DcNP composition effectively targeted lymphatic-vessel-rich 4T1 primary tumors in the mammary fat pads. GT-in-DcNP significantly increased tumor accumulation of both drugs, approximately 10-fold higher than that of the equivalent free-drug combination, resulting in primary tumor regression and restoration of mammary fat pad tissue. For systemic drug exposure, intravenous administration was employed to treat lung-metastatic tumor nodules in a 4T1-bearing mouse model. The PK behavior of multiple GT-in-DcNP compositions was first evaluated in healthy mice, confirming long-acting PK for both drugs across all formulations. In parallel, DcNP was shown to protect gemcitabine from rapid metabolism by cytidine deaminase, which likely contributes to its prolonged systemic exposure. This protective effect also enabled synchronized delivery of both drugs, with approximately 98% in vivo association efficiency of gemcitabine across compositions. Taken together, this dissertation elucidates the critical role of formulation mechanisms in enabling long-acting and synchronized delivery of physicochemical diverse drugs at various ratios using the DcNP platform, as demonstrated by GT-in-DcNP.