FUS is a relatively new technology that is gaining wide clinical acceptance for targeting various tumors within the body (Carpentier et al., 2016, Chen et al., 2020, Idbaih et al., 2019, Mainprize et al., 2019, McMahon et al., 2019, Meng et al., 2019, Park et al., 2021, Sarkaria et al., 2018). FUS therapy using microbubbles (MBs) greatly expands the versatility of this technique. It leverages the mechanical effects of acoustic cavitation to facilitate drug delivery across biological barriers. When exposed to ultrasound, MBs oscillate, undergoing volumetric changes that result in stable or inertial cavitation, depending on the acoustic pressure. In stable cavitation, microbubbles oscillate symmetrically, producing microstreaming and shear stress on nearby cell membranes, which can enhance processes such as endocytosis. In contrast, inertial cavitation occurs at higher acoustic pressures, leading to violent microbubble collapse that generates shock waves, microjets, and high shear forces capable of inducing pore formation in the vasculature, otherwise known as sonoporation. These pores facilitate the uptake of therapeutic agents, such as liposomes, by the targeted tissue (Tu and Yu, 2022, Izadifar et al., 2020, Frinking et al., 2020, Bellary et al., 2020).

When using FUS for drug uncaging, the drug is usually loaded on the surface of MBs (Rastegar et al., 2023, Chapla et al., 2022, Ma et al., 2013). Upon exposure to ultrasound, their volumetric oscillation generates significant shear stress and heat, expediting the release of their cargo into circulation (Honari and Sirsi, 2023).

Currently, most contrast agents used in FUS therapy are biologically inert, primarily responding to the applied ultrasound beam. Recently, we have started exploring biologically active ultrasound contrast agents that respond acoustically to blood oxygen levels, using hemoglobin-based microbubbles (Chaudhary et al., 2024). Utilizing focused ultrasound for drug delivery with these oxygen-sensitive contrast agents offers an opportunity for enhanced precision, potentially improving the differentiation between hypoxic and healthy tissues within the focus of the beam. A method of more broadly targeting tumors and metastatic spread would allow a simpler approach that is more clinically feasible.

Hemoglobin carriers are predominantly employed for oxygen delivery to hypoxic regions (Zhao et al., 2007, Wong and Suslick, 1994), capitalizing on hemoglobin’s inherent function in oxygen transport. However, in select cases, hemoglobin carriers have shown promise in drug delivery (Yang et al., 2018, Zhao et al., 2019) by encapsulating drugs through non-covalent interactions. These approaches have primarily relied on passive targeting strategies, which are inherently reliant on the tumor enhanced permeability and retention (EPR) effects, which is known to be overexaggerated in many small animal models (Danhier, 2016).

Various diseases are characterized by hypoxia, metabolically active regions where oxygen levels are significantly lower than in surrounding healthy tissues. Cancerous tumors, for example, often have hypoxic cores due to rapid cell proliferation outpacing the development of new blood vessels. Similarly, ischemic heart disease and certain neurological disorders, like stroke and neurodegenerative diseases, create localized hypoxia as cells struggle to access sufficient oxygen.

Hemoglobin-based MBs (HbMBs) used as drug carriers could offer a unique advantage for drug delivery in these contexts. By responding to oxygen gradients, HbMBs could preferentially release therapeutic agents in hypoxic, disease-affected tissues, ensuring that treatment reaches the most metabolically active and oxygen-deprived areas. This targeted delivery could improve therapeutic outcomes while minimizing off-target effects, making hemoglobin carriers a valuable tool for treating hypoxia-associated diseases.

Previously, we developed hemoglobin microbubbles (HbMBs) for oxygen biosensing and provided proof of concept for their potential in real-time blood-oxygen-level-dependent (BOLD) imaging (Chaudhary et al., 2024, Rastegar et al., 2025). HbMBs showed significant differences in their acoustic responses in oxygenated (Oxy) versus deoxygenated (Deoxy) environments. This is due to the conformational changes in hemoglobin under these conditions, which significantly alter the elasticity of Hb molecules, resulting in a shift in HbMBs resonate frequency. Consequently, HbMBs exhibit varying acoustic signal intensities depending on their elasticity in oxy or deoxy environments. In this study, for the first time, we report an oxygen-sensitive, hypoxia-targeting drug delivery carrier based on hemoglobin microbubbles (HbMBs). A schematic illustrating this is presented if Fig. 1. HbMBs were conjugated with doxorubicin (DOX)-loaded liposomes (LDOX) and subsequently exposed to US in media with partial oxygen pressures (PO2) of 160, 120, 85, 42, and 5 mmHg. Results demonstrated significantly higher drug release at PO2 of 5 mmHg compared to other Oxy levels.

This study presents a new strategy in FUS-mediated delivery by introducing “biosensitive” microbubbles that could more specifically target metabolically active tissues and limit delivery of toxic drugs to adjacent healthy cells. Overall, this platform technology could radically change how focused ultrasound treatments are performed in order to make them more effective in a wide-range a of cancer and non-cancer diseases.