(L-471) Optimizing the Formulation of Nanobubble-based Therapeutic Delivery Systems

Introduction: Osteoporosis, a prevalent bone disorder, affects hundreds of millions individuals worldwide. This prevalence, alongside the associated financial burden on healthcare institutions and the insufficiency of current osteoporosis therapies emphasizes the urgent need to develop new gene and drug delivery systems to counter the disease. A promising approach involves nanobubbles (NB), whose properties such as small size, ultracontrast in diagnostic imaging, and targeted delivery capabilities, render them an ideal platform. A key aspect of our methodology involves the use of low-intensity pulsed ultrasound (LIPUS), which stimulates the growth and expansion of NB, which introduces another layer of precision control in NB release and efficiency. Human serum albumin (HSA), serving as the encapsulating shell, can influence the size and stability of these NB. Cathepsin K (CTSK) is upregulated by osteoclasts, which digest bone, contributing to the development of osteoporosis when overactive. However, CTSK can be downregulated through the introduction of targeted siRNA, disrupting its activation and effectively ‘knocking down’ the gene. In this study, we aim to develop optimally synthesized NB, fine-tuned by precisely determining the optimal amount of HSA. The efficacy of these NB will be validated using metrics including size, growth rate, and polydispersity indices (PDI). Ultimately, we intend to employ these NB as targeted delivery vehicles for CTSK siRNA to the bone, thereby serving as an effective and non-invasive treatment option for osteoporosis.

Materials and Methods: The study utilized in situ sonochemical synthesis for NB preparation, incorporating 300 μL of perfluorocarbon, 4 mL of phosphate-buffered saline, and varying concentrations of HSA. Our study evaluated the incorporation of different HSA concentrations (20 mg, 40 mg, and 80 mg) to optimize NB parameters. Dynamic light scattering (DLS) measurements were assessed to calculate polydispersity index (PDI) and effective diameter of the NB, indicating size distribution. One NB formulation was synthesized per each HSA concentration, with n=3 replicates for each. The prepared NB were dispersed into 2 mL PBS and diluted approximately 100 times using triple-filtered deionized water to prevent multi-scattering. DLS measurements were taken at room temperature using a 640 nm laser filter, with each experiment running for approximately 2 minutes in the instrument (Brookhaven Instruments). Acoustic induction via low-intensity pulsed ultrasound (LIPUS) was later employed to initiate NB growth and expansion, monitored in real-time via optical imaging microscopy. To prepare for imaging, 20 mg portions of the formed NB pellets were transferred into 2 mL of PBS in 6-well plates. NB were stimulated using LIPUS program at 1 W/cm² intensity for four minutes. Images were captured at key time intervals to capture critical events such as NB growth and rupturing; these images were organized into a timelapse sequence. Furthermore, images were used to evaluate size parameters of NB (n=100 NB per image slice), and to approximate rate of expansion over a total 2 hour period post-LIPUS stimulation via the ImageJ software. Statistical significance was determined as p< 0.05.

Results, Conclusions, and Discussions: DLS measurement analysis (n=5 NB samples each) demonstrated NB synthesized with 40 mg HSA had the lowest PDI (0.194), indicating a monodisperse size distribution, critical for gene delivery as it implies a more controlled, reproducible, and homogenous delivery. In comparison, the 80 mg HSA concentration had a PDI of 0.289, while 20 mg HSA concentration had a PDI of 0.309. Additionally, NB size over a 2-hour period post-low-intensity pulsed ultrasound (LIPUS) stimulation was confirmed utilizing a 4X AmScope microscope. In 20, 40, and 80 mg HSA NB comparisons, all 3 sample NB expanded and grew over time, with measurements at 1 min post-LIPUS (n=50 for each group) demonstrating diameters of 178±30 μm (20 mg HSA), 99±25 μm (40 mg HSA), and 73±30 μm (80 mg HSA). After 120 minutes, NB diameters expanded to 628±40 μm, 531±25 μm, and 331±30 μm for 20 mg, 40 mg, and 80 mg HSA, respectively.

Uniform size distribution and stability, represented by a low PDI as seen in 40 mg HSA display PDI of 0.194, are critical for a controlled biodistribution and systemic clearance in therapeutic and diagnostic (termed "theranostic") applications. In contrast, NB with 20 mg HSA showed rapid growth but risked premature release with its largest diameter expansions. 80 mg HSA NB demonstrated slower growth and diameters, perhaps attributable to a larger quantity of HSA forming a robust, overly-stabilizing outer coating, resulting in a less US-responsive NB that limits gene release efficiency.

Our findings highlight the influence of HSA concentration on NB properties. Furthermore, it indicates that 40 mg HSA concentration yields the most stable and uniform NB. Thus, it was selected as the optimal amount of HSA for NB synthesis for systemic circulation and controlled gene release. Despite the limitations of the other tested HSA parameters, unique properties of the 20 mg and 80 mg HSA NB may prove useful in other applications, such as wastewater management. Ultimately, our study lays a foundation for further research to optimize NB for targeted osteoporosis CTSK siRNA gene delivery therapies, and beyond.

Acknowledgements (Optional): We acknowledge the support received from UCF Department of Medicine Seed Grant and Research Foundation Gap Fund. We also appreciate Dr. Sara Tabandeh for providing the required training for Dynamic Light Scattering.