PEI-Coated Microbubble Attachment to Neutrophils: Potential for Radiation Force Assisted Delivery into Tissue

Immunotherapies have advanced cancer treatment; however, their clinical efficacy remains limited for solid tumors due to challenges associated with effectively directing immune cells into the complex tumor microenvironment. Recent developments in Ultrasound Contrast Agent (UCA -- also known as "microbubble") technology have provided novel opportunities to enhance targeted therapeutic delivery. In this study, we introduce an innovative approach of leveraging microbubbles to enhance immune cell targeting by directly attaching microbubbles to immune cells, enabling the targeted delivery and localization of immune cells into solid tumors using radiation force ultrasound (US) application. To create novel microbubble-immune cell conjugates, we created polyethyleneimine (PEI) coated microbubbles and attached them to differentiated HL-60 (dHL-60) cells. These positively charged PEI microbubbles were formulated using azide-DBCO click chemistry between DBCO-labeled microbubbles and the azide functional groups on the PEI polymer. Following this step, we utilized electrostatic interactions to attach our positively charged PEI microbubbles to our negatively charged dHL-60 cells. We conducted viability experiments to assess the compatibility of these designs and verified that cell viability remained greater than 88% four hours after the conjugation process for different ratios of dHL-60 cells to PEI microbubbles. We used microfluidic chemotaxis platforms to quantify the microbubble-conjugated dHL-60 cell migratory behavior, examining parameters including migration velocity and percentage. Additionally, we investigated the impact of ultrasound power on primary human neutrophils to validate the functional responsiveness of these physiologically relevant immune cells. Here we demonstrated the possibility of ultrasound-responsive immune cell constructs as a targeted strategy without loss of function in migration capabilities. The novel PEI microbubble and immune cell conjugates reported in this work will be used to improve future immunotherapy techniques.