Development of Novel Polymer Ultrasound Contrast Agents Incorporating Nested Carbon Nanodots
Authors:   , Shirley Matthew , Wheatley Margaret A. 
Published: January 2025

In 2024, a total of over 2 million people in the United States are expected to be newly diagnosed with cancer, and over 600,000 people in the country are expected to succumb to the disease. These facts, along with the recent $150 million reinvestment in the Cancer Moonshot initiative, highlight the urgent necessity for novel cancer detection and treatment strategies. Great progress has been made over the years; however, the systemic delivery of non-specific and toxic chemotherapeutic regimens is still a hallmark of many current treatment options in use today. This lack of therapeutic effectiveness is compounded by numerous other factors, including dosage restrictions to limit this toxicity, high inter-tumoral fluid pressures, blood flow disturbances, and a reliance on passive targeting. Several theranostic strategies have been investigated, including the usage of microbubbles, or ultrasound contrast agents (UCAs). Microbubble-mediated ultrasound therapy has gained significant attention due to its non-invasive nature and targeted delivery potential. The Microencapsulation Lab has developed a drug-loaded polymer microbubble that can be used as a UCA, and the polylactic acid (PLA) microbubbles have the robust capability of being able to load nanoparticles in the shell, as well. However, current polymer microbubbles still face limitations in terms of their functionality, leaving room for improvement, particularly in imaging performance and the functional versatility of the platform. To date, the FDA has not approved any polymeric agents for commercial use. In this context, the integration of carbon nanodots (CNDs) with polymer microbubbles presents an exciting avenue for the improvement of microbubbles. CNDs are emerging nanoscale (<10 nm), photoluminescent particles with unique properties that exhibit drug loading and delivery capabilities, including rapid cellular uptake and strong biological imaging potential. CNDs have been proven to exhibit reliable photoluminescence and to be strongly emissive. Economically, CNDs are also cost-effective to produce and can be synthesized from a wide variety of carbon sources in minimal steps. Encapsulation of CNDs within the polymer shell of microbubbles can provide multifunctionality, combining the fluorescent visualization and drug-retaining capabilities of CNDs with the drug delivery and real-time ultrasound imaging capabilities of microbubbles. This dissertation describes a research effort to develop a CND-loaded microbubble to enhance the theranostic capabilities of the UCA platform. By encapsulating CNDs within the PLA microbubbles, we hypothesize that the resulting microbubble system will display improved imaging capabilities, allow tracking of collapsed polymer fragments and shells, and potentially improve the loading capabilities of drug, all while maintaining the vital acoustic properties of a functional UCA. To our knowledge, this is the first study to explore incorporating CNDs into a polymer microbubble shell. This study improves upon previous research by incorporating all low to non-toxic carriers while potentially keeping the drug delivery platform clinically relevant. To establish a proof-of-concept CND-loaded polymer UCA, carbon quantum dots (CQDs) were incorporated into the PLA microbubble water/oil/water emulsion fabrication process. This integration led to enhanced acoustic properties, with CQD-loaded microbubbles exhibiting a ~10% improved acoustic enhancement and a >50% improved acoustic stability compared to unloaded microbubbles. In addition to this, the CQDs did not significantly change the physical characteristics of the UCA. Although the presence of CQD in the polymer shell was confirmed through TEM indirectly, confocal microscopy was needed to detect visual fluorescence. To further establish the CND-microbubble loading capabilities, three different graphene quantum dots (GQDs) were compared by organic loading onto the UCA. These unmodified (U-), carboxylated (C-), and aminated GQDs (A-GQDs) affected the UCA in a variety of ways. All UCA experimental groups maintained vital acoustic properties. More uniquely, the U-GQDs increased the microbubble concentration and acoustic enhancement to the highest of the group while also imbuing fluorescence on the shell. The C-GQDs decreased the size and enhancement, and did not exhibit additional fluorescence. The A-GQDs maintained improved physical and acoustic microbubble characteristics compared to the unloaded control, and they exhibited the most advantageous fluorescence and loading capabilities. To continue expanding the platform capabilities, the method of loading A-GQDs was then studied. Three different loading weights were evaluated in two different loading conditions: organic loading or aqueous loading. All A-GQD loaded UCA groups performed similarly except in a couple of areas. The 1wt% loaded A-GQD groups significantly increased particle counts in the microbubble size range, which decreased as loading weight went up, and scanning electron microscopy ultimately showed that 1wt% loading contains non-bubble PLA material while 5wt% aqueous loading of A-GQDs appears to stabilize uniform bubble formation. In terms of nanoparticle loading efficiency, the aqueous loadings consistently showed greater loading efficiencies over the equivalent organic loading group, with 3wt% having the highest efficiency. Co-loading of A-GQD and doxorubicin (Dox) was then examined. This revealed lackluster results, showing barely any microbubble formation, let alone drug-nanoparticle-loaded microbubbles. However, pivoting to examining Dox loading on C‑GQDs revealed that Dox + C-GQD-loaded UCAs made intact microbubbles and improved acoustic behavior over unloaded or Dox-loaded UCAs individually, while also improving the drug loading of Dox compared to Dox alone by ~150%. Overall, this work represents a step towards developing multifunctional theranostic agents, maintaining the acoustic and fluorescent properties of the CND-MBs while incorporating a chemotherapeutic agent. This study contributes to the field of nanoparticle-loaded UCAs and opens a door for future research toward applications in agile, targeted cancer therapies and diagnostic imaging.