Redefining Nanomedicine Beyond One-Size-Fits-All: Uncovering Mechanisms of Sex Differences and Female Life Stages in Drug Delivery - Project Summary Historically, women's health has been significantly under-researched and underfunded, contributing to a profound health gap that equates to 75 million years of life lost globally. Nanomedicine presents a promising solution to addressing this gap, with its ability to precisely target tissues, bypass biological barriers, and control therapeutic release while minimizing toxicity. However, like much of medicine, nanomedicine design has been approached through a one-size-fits-all lens, failing to address variations in biology and nanoparticle (NP) efficacy across diverse patient populations. This absence of tailored design likely contributes to the limited clinical translation of nanomedicines, despite its potential to revolutionize medicine. The proposed research seeks to address critical gaps in our understanding of sex and female life stages in nanomedicine delivery. While previous research has shown that NPs function differently in males and females, the underlying mechanisms are not well understood. Key Question 1 aims to uncover these mechanisms and identify the key factors that impact NP delivery and efficacy by systematically evaluating NP performance across cellular (NP uptake), microenvironmental (protein corona and mucus transport), and systemic (circulation, stability, immune response, biodistribution) levels. For each of these levels, mechanisms driving sex-dependent differences will be investigated. Additionally, this research will go beyond treating sex as binary, considering it as a spectrum that intersects with age and hormone levels in the female body, marking one of the first studies to examine how menstrual cycle phase, hysterectomy, and menopause affect NP protein corona formation, transport, and biodistribution. Importantly, our work will utilize a library of clinically relevant lipid- and polymer-based NPs with precisely controlled size and charge properties, addressing a key gap in limited studies that have explored sex differences in NP delivery but overlooked the importance of these tailored NP characteristics. Moreover, vaginal delivery, which offers transformative potential for localized and systemic treatment, remains underexplored in nanomedicine despite its advantages for women-specific diseases. Key Question 2 will investigate the use of thermo- and enzyme-responsive hydrogels to optimize vaginal NP delivery systems, enhancing precise and effective drug targeting. Vaginal delivery has primarily been evaluated in the context of sexual and reproductive health conditions, but its broader applications, particularly with clinically relevant NPs, remain largely unexplored. This research will compare vaginal delivery of NPs with traditional administration routes such as intraperitoneal and intravenous delivery, seeking to uncover its potential for targeting both reproductive and non-reproductive organs. Ultimately, this research aims to shift drug delivery from one-size-fits- all therapies to personalized nanomedicines that address the diverse needs of patient populations, ultimately advancing more equitable healthcare outcomes.