Transmembrane Cell Signaling by Targeted Ultrasound Contrast Agents in Cancer Therapy
Authors:   , Jablonowski Lauren Jo , Wheatley Margaret A. 
Published: July 2021

In 2016, over 1.68 million people in the United States are expected to be diagnosed with cancer, with approximately 35% of these cases resulting in death making cancer the second most common cause of death in the US [1]. Specifically, the American Cancer Society predicts that approximately 250,000 new breast cancer cases will be diagnosed in 2016, with approximately 16% of these cases resulting in death [1]. While significant progress has been made in the field of cancer treatment, many therapy regimes still rely heavily on the systemic administration of toxic chemotherapeutic agents. Most clinically used chemotherapeutic drugs are non-specific and as a result are highly toxic to both cancerous and non-cancerous tissues, leading to dangerous side effects and overall low treatment efficacy. Ultrasound contrast agents (UCA) have emerged as a drug delivery vehicle to overcome these challenges, consisting of a small (<6[mu]m) gas bubble stabilized within a lipid, protein, or polymer microcapsule shell that can be injected intravenously and pass freely through the capillaries. Upon entering the focused ultrasound beam, the UCA gas core rapidly expands and contracts, a phenomenon known as cavitation, and ultimately ruptures producing drug-loaded particles (up to 400nm) that can exit the vasculature, embed in the target tissue, and provide localized drug release. Our lab has previously developed hollow polymeric microspheres that encapsulated doxorubicin (Dox) within the polylactic acid (PLA) shell [2, 3]. These Dox-loaded UCA have been shown to exhibit high echogenicity, and also shatter into nanoshards (n-Sh) of less than 400nm, when exposed to ultrasound [4]. When evaluated in an in vivo rat hepatocellular carcinoma model, we found these n-Sh were able to effectively exit the vasculature and deliver Dox to tumor tissue [5]. However, elevated Dox levels were also observed in the healthy liver and spleen tissues, suggesting immune recognition and uptake of circulating agents and n-Sh. Here we describe the development of non-immunogenic, functionalized PLA UCA for use in targeted breast cancer therapy. We believe that the use of a targeting ligand in conjunction with polyethylene glycol (PEG) will help to shield the UCA from immune recognition and improve the treatment efficacy at the target site. For ultrasound (US) imaging with polymeric contrast agents, it is necessary to modify the shell to create "stealth" microbubbles but without these modifications sacrificing the agent's ability to interact with the focused US beam. We hypothesize that addition of the classic immune shielding molecule PEG to a polylactide (PLA) microbubble shell will affect the acoustic and physical properties of the resulting agents. In an effort to determine the best formulation to achieve a balance between stealth and acoustic activity, we compared two PEGylation techniques; addition of increasing amounts of PEG-PLA copolymer and employing incorporation of a PEG lipid (LipidPEG) into the shell. Loss of acoustic enhancement occurred in a dose-dependent manner for both types of PEGylated agents (loss of signal occurred at >5 wt% PEG-PLA and >1 wt% LipidPEG), while immune activation was also reduced in a dose-dependent manner for the PEG-PLA agents. This study shows that the balance between acoustic behavior and improved immune avoidance was scalable and successful to different degrees with both PEGylation methods, and was best achieved using for PEG-PLA at 5 wt% and for LipidPEG at 1 wt%. The added advantage to the use of the targeting ligand, specifically tumor necrosis factor-related apoptosis inducing ligand (TRAIL), is that it also induces tumor cell death upon binding to the cell surface receptors DR4 and DR5 and initiating a transmembrane apoptosis signal. Healthy cells possess decoy receptors (DcR1 and DcR2) that cannot process the apoptotic signal, therefore being protected from non-specific binding. Additionally, the UCA were designed to co-encapsulate the chemotherapeutic drug doxorubicin (Dox) that can be released from the polymer shell in response to US focused at the tumor site, shielding healthy tissues from the toxic substance while also increasing the potency and efficiency of treatment to the tumor tissue. The ability of the ligand to cause cell death was tested against ligand-sensitive MDA-MB-231 human breast adenocarcinoma cells and ligand-resistant MCF7 human breast adenocarcinoma cells, comparing the efficacy of the microbubble formulations. It is believed that co-administration of Dox to cancer cells that are normally resistant to TRAIL increases the expression of death receptors on the cell surface, sensitizing the cell to TRAIL and improving its efficacy. For the MDA-MB-231 cells, cell viability was reduced by approximately 25-50% (52.84±11.65% to 76.34±3.25% live cells) upon incubation with the TRAIL-ligated US-generated nanoshards, and was further reduced to approximately 40-80% (20.32±6.91% to 64.19±2.68% live cells) with the addition of doxorubicin compared to TRAIL alone. TRAIL-resistant MCF7 cells showed little apoptotic response to TRAIL-ligated nanoshards (93.44±2.88% to 98.18±1.04% live cells); however, co-administration of doxorubicin increased apoptosis and reduced cell viability (37.37±5.39% to 67.78±3.98% live cells), supporting the sensitization effect of the drug. Healthy MCF-12A human breast epithelial cells were also tested, to confirm the selective targeting and apoptotic activity of TRAIL to cancer cells and not healthy cells. These cells exhibited from 90.64±2.54% to 97.46±0.62% cell viability when incubated with TRAIL-ligated nanoshards, confirming their insensitivity to TRAIL-induced death. However, cell viability was greatly decreased (44.73±15.26% to 68.79±6.89% live cells) when also exposed to doxorubicin, demonstrating the toxic effects of the chemotherapeutic agent on surrounding healthy cells during systemic treatment. Overall, this work has resulted in the production of effective ultrasound-triggered, non-immunogenic, targeted drug delivery agents for potential use in cancer therapy. This platform has many advantages over the systemic administration of chemotherapeutic drugs, and represents a promising treatment to better serve the population with breast cancer, and solid cancerous tumors as a whole.