Experimental Study on the Compatibility and Characteristics of a Dual-Target Microbubble Loaded with Anti-miR-33

Chen Yuan,1,* Yanhong Li,1,* Liyun Liu,1 Baihetiya Tayier,1 Lingjie Yang,1 Lina Guan,1– 3 Yuming Mu1– 3 1Department of Echocardiography, First Affiliated Hospital of Xinjiang Medical University, Urumqi, 830011, People’s Republic of China; 2State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, First Affiliated Hospital of Xinjiang Medical University, Urumqi, People’s Republic of China; 3Xinjiang Key Laboratory of Medical Animal Model Research, Clinical Medical Research Institute of First Affiliated Hospital of Xinjiang Medical University, Urumqi, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yuming Mu; Lina GuanDepartment of Echocardiography, The First Affiliated Hospital of Xinjiang Medical University, 137 Liyushan Road, Ürümqi, Xinjiang Uyghur, Autonomous Region, 830054, People’s Republic of ChinaEmail mym1234@126.com; sanjin_lsx@163.comObjective: To prepare a new type of dual-target microbubble loaded with anti-miR-33 (ANM33).Methods: Carrier core nanobubbles (NBs) were prepared by thin film hydration, and microbubbles loaded with PM1 (PCNBs) were prepared by grafting DSPE-PEG2000-maleimide-PM1 onto the NB surface. ANM33 was connected via electrostatic adsorption and covalent bonding, and hyaluronic acid (HA) was covalently connected. PM1 and HA were the targets, and ANM33 was the intervention drug. To evaluate the general physical and chemical properties of the prepared dual-target microbubbles loaded with ANM33 (HA-PANBs), we observed their morphology, particle size and surface potential while monitoring their stability and in vitro imaging ability, evaluated their toxic effect on cells and verified their ability to target cells.Results: HA-PANBs had a regular morphology and good stability. The average particle size measured by a Malvern potentiometer was 1421.75± 163.23 nm, and the average surface potential was − 5.51± 1.87 mV. PM1 and ANM33 were effectively connected to the NBs. The PM1, ANM33, and HA binding reached 89.0± 1.1%, 65.02± 5.0%, and 61.4± 3.5%, respectively, and the maximum binding reached 2 μg, 5 μg, and 7 μg/108 microbubbles, respectively. HA-PANBs had no obvious toxic effects on cells, and their ability to continuously enhance imaging in vitro persisted for more than 15 minutes, obviously targeting foam cells in the early stage of AS.Conclusion: HA-PANBs are ideal ultrasound contrast agents. The successful, firm connection of PM1 and HA to the NBs significantly increased the amount of carried ANM33. When microbubbles prepared with 2:4:7 PM1:ANM33:HA were used as a contrast agent, they had a high ANM33 carrying capacity, stable physical properties, and significantly enhanced imaging and targeting of foam cells in the early stage of AS.Keywords: ultrasound microbubbles, molecular targeting, dual targeting, atherosclerosis