Development of nano-sized polymeric ultrasound contrast agents for cancer diagnosis and therapy
Authors:   , Kwon Seunglee , Wheatley Margaret A. 
Published: July 2021

Our overall target is cancer. Among several cancer diagnostic imaging modalities, ultrasound imaging has attracted much attention due to its noninvasiveness, safety, portability, relatively low cost and real-time 3D imaging opportunity. Currently, therapeutic contrast agents are under development to perform as a targeted drug delivery carrier while improving the ultrasound image quality of the targeted region. Modern ultrasound contrast agents (UCA) primarily comprise stabilized microbubble formulations that circulate in the intravascular compartment. In order for the contrast agents to achieve effective target specific detection and delivery of chemotherapeutic drugs, it is necessary for the agents to reach to the target cancer cells where the drug needs to be released to act. Nanotechnology may hold the key to accomplish this since nanoparticles (NP) below 400 nm would pass through the leaky angiogenic vessels, accumulate within tumor interstitum and interact directly with the cancer cells. Also, the size range of 120 to 200 nm in diameter is generally known to have longer circulation time in blood vessel by substantially avoiding particle trapping via reticuloendothelial system (RES) and renal clearance. The challenge is now to overcome inherent weak echogenicity of the NP. A hollow structure created by removal of a porogen, stabilized gas molecules, and highly concentrated deposition were found to be the best strategies to improve the backscattering enhancement of the UCA. Combination of these approaches was attempted to create nano-sized UCA (n-UCA), herein. In this research, a poly (D,L-lactic acid) (PLA) based n-UCA was first produced by modifying a single emulsion-solvent diffusion method to incorporate hydrophobic sublimable porogen such as camphor. The porogen was added to make the NP hollow upon removal, enabling gas introduction, which offers echogenicity to the particles. A polymeric platform was chosen since it bears the advantages of being more stable and degradable in a more controlled manner than a lipid platform, which was primarily used as a microbubble stabilizing shell, to date. Of polymers, PLA was selected because it is biocompatible, biodegradable, and FDA-approved. Dynamic light scattering (DLS) and microscopic results demonstrated that the NP have approximately 200 nm size with a spherical shape and unimodal distribution. The dispersion of hollow NP containing perfluorocarbon (PFC) gas showed 14.1 ± 2.3 dB enhancements in backscattering with a dose of 0.2 mg/ml at 5 MHz frequency, which is within medical ultrasound imaging frequency range, 2-15 MHz. The achieved enhancement was not the highest that is possible to obtain for in vivo imaging. Combining this NP with the minimal amount of highly echogenic micro-sized PLA based UCA, which was already developed in our laboratory, would be the way to increase overall agent capability. This is because in the case of the combined platform, the advantage lies in getting the maximum benefit from both NP and micro-sized particles (MP) for cancer targeted drug delivery (nano) and ultrasound imaging (micron), respectively. A single process to prepare bimodal distributed PLA particles (BP) involving both micro-sized and nano-sized populations was also optimized by adapting a salting out method to load porogens. We were also motivated to create echogenic nanomicelle (NM) based on mPEG-PLA block copolymer due to a novelty of the method and expected benefits of reducing nonspecific cellular uptake and increasing blood circulation time from the PEG outer layer of the micelle in intravascular administration. The micelle forming process also can avoid the use of particle stabilizer, poly (vinyl alcohol) (PVA), which is known to possibly block surface functional groups which are used to conjugate a targeting moiety to the bubbles. Co-solvent lyophilization method enabled generation of around 200 nm sized micelles based on the block copolymers. However, our echogenicity enhancing strategy with porogen and gas was not successful for improving the acoustic property of the NM, which we reported in this thesis. Lastly an ovarian cancer (OC) targeted PLA NP was produced with a hope to enable early detection of OC by extravasating the tumor vasculature and adhering to the OC molecular marker, CA125 of the OC cells. To target CA 125, anti-CA125 antibody, OC125, was conjugated to the NP. Under the fluorescent microscope and confocal microscope, the cellular destination of the OC125-NP conjugate within cytoplasm was visualized with an aid of red fluorescent dye, nile red (NR), which is incorporated into the particles prior to the antibody (Ab) conjugation. Due to the larger surface area/ volume ratio, excellent conjugation efficiency (above 90%) could be achieved with NP over MP (~70%). Unlike OC125-MP conjugate, which was attached to the CA125 positive cell surface, the nile red loaded OC 125-NP (NR-NP-Ab) was uptaken by cells. Thus, the NR-NP-Ab is not expected to form a deposition layer which has a feasibility to improve backscattering of the targeted region. Nevertheless this feature would be beneficial for intracellular drug delivery. As an additional first attempt toward therapeutic application doxorubicin, anti-cancer drug, was loaded onto NP and BP and compared with MP. Again, the larger surface/ volume ratio of NP allowed the higher drug loading efficiency via surface absorption (76.3 ± 6.8 % for NP, 31.4 ± 8.7 % for BP, and 14.8 ± 0.6 % for MP). It should be noted that these works were very preliminary, thus much additional work remains on assessing detailed optimization and method development focusing on n-UCA. In conclusion, this study presents pioneering work to develop ultrasound imaging using nanobiotechnolgy. Furthermore, the developed BP and mixture of MP and NP or MP and NM would be good candidates for integrating several therapeutic application areas such as site-specific targeting and eventually intracellular drug delivery to treat cancer along with the ultrasound imaging.