 of incubation with PAI-1, MMB-SiO2-tPA still maintained 36% of the tPA activity, which was higher than 16 and 8% obtained by SiO2-tPA and native tPA, respectively (Fig. 2H). It is worth noting that the close packing of shelled nanoparticles on the microbubble surface prevented the release of tPA from silica nanocarriers without an ultrasound trigger, resulting in the higher retained activity of MMB-SiO2-tPA than that of SiO2-tPA.
This microbubble-based drug delivery system is expected to release the drug upon the ultrasound stimulation. Traditionally, most microbubbles are stabilized by a rigid shell formed by polymer, silica, or protein (21, 23). The release of cargos depends on the bubble inertial expansion and explosion, a phenomenon called cavitation that usually requires high acoustic-driven force (i.e., high-intensity ultrasound) (24). To reduce the risk of tissue injury in cavitation, drug release by stable microbubble oscillations (fig. S3) that activated by low-intensity ultrasound is an alternative (20, 25, 26).
We first validated the response of MMB-SiO2-tPA toward the magnetic guidance in an in vitro vessel system. The MMB-SiO2-tPA were dissolved in cell culture medium and pumped by a syringe into a polyethylene vessel (diameter of 3 mm) with a speed (1.2 cm s1) that mimics the blood flow. When a magnet was placed adjacent to the vessel, MMB-SiO2-tPA accumulated to the magnet in approximately 2 s (fig. S4). Once the magnet was removed, the accumulated MMB-SiO2-tPA swiftly dispersed and circulated with the liquid flow without noticeable attachment to the vessel wall.
The thrombolysis efficacy was further evaluated in the ex vivo blood clots, which were prepared by fresh mice blood and thrombin (Fig. 5A). With dissolution, the clots in tubes shrank, and the supernatants became red. As expected, clots treated by saline barely lysed in a period of 12 hours, suggesting the stability of prepared clots. When clots were treated with native tPA, SiO2-tPA, or MMB-SiO2-tPA, all clots lysed stepwise into smaller sizes, while the supernatants gradually changed from colorless into blood red (Fig. 5B). The black color presented in MMB-SiO2-tPAtreated group was generated by the released iron oxide nanoparticles. The dissolution efficiencies were quantified by measuring the mass loss of the clots at 3 and 12 hours after treatments. During the first 3 hours (Fig. 5C), clots treated by MMB-SiO2-tPA exhibited similar dissolution efficiency (approximately 32%) to those by native tPA (approximately 27%), which was higher than those obtained by SiO2-tPA or saline treatments (17 and 7%, respectively). After 12 hours (Fig. 5D), MMB-SiO2-tPA treatment achieved the highest dissolution efficiency, which was approximately 93%, compared to those obtained by native tPA, SiO2-tPA, or saline treatments (68, 51, and 16%, respectively). The amounts of hemoglobin (released during clot lysis) in the supernatants at 12 hours after treatments exhibited similar trends to the dissolution efficiencies, confirming the results obtained in the ex vivo experiments. As this experiment was performed in a static situation without blood flow, the improvement of dissolution efficiency in the MMB-SiO2-tPAtreated group might be attributed to the enhanced penetration and retained activity of tPA.
(A) Schematic illustration of the blood clot dissolution treatment process under the magnetic field combined with low-intensity ultrasound. (B) Representative images of the thrombolysis processes at 0, 3, 6, 9, and 12 hours after being treated by saline, native tPA, SiO2-tPA, and MMB-SiO2-tPA, respectively. (C) Quantification of the dissolution efficiency by measuring the mass loss of the blood clot at 3 hours after treatments. (D) Quantification of the dissolution efficiency by measuring the mass loss of the blood clot at 12 hours after treatments. (E) Absorbance values ( = 540 nm) of the supernatants at 12 hours after treatments; n = 5; *P < 0.05, **P < 0.01, and ***P < 0.001.
To investigate whether the clot-penetrating strategy was applicable to in vivo animal models, we performed different treatments to the femoral vein thrombi of a mouse model. Male C57/BL6J mice were pretreated with FeCl3 for infiltration to form the clots, then were injected with MMB-SiO2-tPA through the tail vein, and were treated with magnetic targeting and the low-intensity ultrasound in sequence (Fig. 6A). Control groups included mice injected with saline, native tPA, and SiO2-tPA, respectively. The images of the thrombi were recorded every 3 hours before the mice were sacrificed at 12 hours after treatments. As shown in Fig. 6B, the femoral vein exhibited dark areas where FeCl3 filter paper was placed, indicating the successful formation of the blood clots. The dark areas of the femoral vein treated by saline became longer with time, suggesting the progression of venous thrombosis. When native tPA were intravenously injected, the dark areas shrank in size and became shallow, indicating that clot lysis happened gradually with time. By this dosage of native tPA (100 l, 10 g ml1), the blocked veins were not thoroughly recanalized, as shallow shadows still presented at 3 and 12 hours after treatment. Although SiO2-tPA holds the ability to protect native tPA against its inhibitors in blood, SiO2-tPA treatment only achieved similar thrombolysis efficacy to that of native tPA treatment, suggesting the low delivery efficiency of SiO2-tPA to clots. With the equivalent amount of tPA, the dark areas of MMB-SiO2-tPAtreated veins faded completely even at 3 hours after treatment. The histological results (Fig. 6C) of the femoral veins were in good agreement with the results in Fig. 6B. Ideally, the thrombus is considered to be a cylinder, and the quantification of the volume of the clots reveals the thrombolysis efficacy. Alternatively, quantification of the thrombus area (% vein lumen) by measuring the cross-sectional areas of the clots and the veins in the histological images provides a rough estimate of the thrombosis area (28). Still, MMB-SiO2-tPA treatment achieved the optimal thrombolysis efficacy, resulting in the least thrombus area (approximately 13%), whereas the percentages were 66, 40, and 50% for treatments with saline, native tPA, or SiO2-tPA, respectively (Fig. 6D). It is worth noting that, according to the histological analysis (Fig. 6C and fig. S7), two of four femoral veins treated by MMB-SiO2-tPA achieved complete recanalization. Furthermore, the improved penetration of released nanoparticles was evidenced by the histological images of the clots (Fig. 6E). The shelled nanoparticles (black dots) mainly attached to the periphery of the clots without an ultrasound trigger. When triggered by low-intensity ultrasound, shelled nanoparticles were released and penetrated into the interior of the clots, resulting in a relatively homogenous distribution of nanoparticles within the clots.
(A) Schematic illustration of the treatment procedures of a femoral vein thrombosis mouse model. (B) Representative images of thrombolysis evaluation after treatment with saline, native tPA, SiO2-tPA, and MMB-SiO2-tPA, respectively. The white arrows indicate the inducted thrombi (n = 4). (C) Representative histological analysis of the femoral vein after treatment with saline, native tPA, SiO2-tPA, and MMB-SiO2-tPA for 12 hours, respectively (n = 4). Scale bar, 50 m. (D) Quantification of the thrombus area (% vein lumen) in femoral vein in different treatment groups (n = 4). (E) Representative histological analysis of the femoral vein after administration and magnetic targeting of MMB-SiO2-tPA with or without low-intensity ultrasound. The red arrow indicates the enrichment of MMB-SiO2-tPA, and the red circles indicate the MMB-SiO2-tPA with an intact structure. Scale bars, 50 m.
To evaluate the safety of the precision delivery strategy in vivo, MMB-SiO2-tPA were intravenously administrated to the mice, and the major organs were collected. As shown in the histological images (fig. S8A), no obvious organ damage or inflammatory lesion appeared in both short-term (i.e., 1 day) and long-term (i.e., 7 days) periods. Meanwhile, serum biochemistry assay and complete blood panel tests were studied by comparison between healthy mice and MMB-SiO2-tPAinjected mice (after 7 days) (fig. S9). The results showed no significant differences in all measurement indicators between the two groups, suggesting the good biocompatibility of MMB-SiO2-tPA. The SiO2 nanoparticles were mainly accumulated in the liver and kidney after 24 hours after injection, while the distribution mainly in the spleen was observed for iron oxide nanoparticles (fig. S10). The biodistribution of MMB-SiO2-tPA in mice suggested their clearance by the reticuloendothelial system.
Last, but not the least, the safety assessment of ultrasound intensity on mice vascular injury was performed. The applied high-intensity ultrasound (above 0.04 MPa, i