Accelerate thrombolysis through shelled nanobubbles using a precise, clot-permeable drug delivery strategy

Conventional thrombolytic drugs such as tissue plasminogen activator (tPA) for vascular obstruction face problems such as low bioavailability, off-target side effects, and restricted thrombus penetration, leading to delayed recanalization. We hypothesize that these challenges can be solved by targeted and controlled thrombolytic drugs or precise drug delivery. A porous magnetic microbubble platform was developed to formulate tPA. The system can maintain tPA activity during the cycle, guide it to the thrombus through electromagnetic induction, and then activate it remotely to release the drug. Ultrasound stimulation also improves the penetration of the drug into the thrombus. In the mouse model of venous thrombosis, the residual thrombus was reduced by 67.5% compared with traditional tPA injection. Ultrasonic penetration of tPA thrombus is as high as several hundred microns. This strategy not only improves the therapeutic effect, but also accelerates the rate of hemolysis, making it very promising in time-critical thrombolytic therapy.
Vascular obstruction partially or completely blocks the blood flow in the blood vessels, usually leading to life-threatening diseases such as coronary artery infarction, ischemic stroke and pulmonary embolism (1-3). Although thrombolytic drugs (such as tissue plasminogen activator (tPA)) through systemic administration or catheter placement have greatly improved the survival rate and quality of life of patients, there are still low bioavailability such as tPA , Poor delivery efficiency and thrombosis. The resistance of fibrin and platelet-rich thrombi to thrombolytic drugs leads to gradual or slow recanalization (4-7).
The delivery of targeted and controlled thrombolytic drugs has been proposed to meet the challenges, aiming to improve the bioavailability of tPA, targeted delivery and acceleration of clot dissolution (8-11). For example, a shear-responsive carrier for narrowing or blocking blood vessels can reduce the required tPA dose and minimize side effects (12). The rotating magnetic nanomotor improves the transport of tPA at the interface of blood and blood clot, thereby accelerating the rate of hemolysis and increasing the thrombolytic effect (13). Through cavitation or acoustically driven diffusion effects, triggering the acoustically triggered release of tPA from tPA-loaded echogenic liposomes can enhance thrombolytic activity (14, 15). Although these advances are encouraging, they only partially solve the above-mentioned challenges and have not yet been translated into clinical practice.
In this process, there are three keys, namely maintaining drug activity, selected accumulation in the blood clot, and diffusion/permeation throughout the blood clot tissue (16-18). Although shear-activated nanotherapy is expected to improve the delivery efficiency of blood clots, tPA loaded by surface binding is often inactivated by inhibitors in the blood (12). Ultrasound can trigger the release of tPA from tPA-loaded echogenic liposomes directly to the clot site visualized by ultrasound imaging. However, due to the lack of targeted strategies, it is limited (14, 15). The thrombolytic efficacy of these methods is also challenged by the limited penetration of the released tPA clot. Magnetically targeted delivery of tPA and mass transfer (diffusion) enhanced by tPA rotation are achieved through magnetic porous micromotors (19). This method solves the above-mentioned challenges and improves the therapeutic efficacy of blood clots in midbrain mice, thereby representing a potential solution for accelerated thrombolysis. Despite the optimistic outlook, the incentive to release tPA from the carrier will reduce unnecessary leakage during the cycle. At the same time, promoting the rapid penetration of tPA during the treatment process will initiate an enlarged interaction area between tPA and blood clots, which may further enhance the thrombolytic effect.
To address this unmet need, we have proposed a precise delivery strategy using magnetic targeting and ultrasound triggered release. Specifically, this strategy uses a multifunctional nanosystem that responds to magnetic fields and ultrasound by coordinating the different functions of its components (Figure 1A). The system stably maintains tPA activity during the cycle. When guided by a magnet, it directly targets the thrombus, which is then remotely activated using low-intensity ultrasound to release the drug. Ultrasound stimulation can also quickly improve the penetration of the drug into thrombus tissue within a few minutes.
(A) Schematic diagram of targeted delivery and controlled release of thrombolysis. (B) Schematic diagram of the synthesis of nano-drug shell microbubbles (ie MMB-SiO2-tPA).
As a proof of concept, we developed a nanoparticle shell microbubble (MMB-SiO2-tPA) for targeted delivery of tPA to blood clots. Microbubbles are made by self-assembly of nanoparticles at the liquid-gas interface (Figure 1B). The prepared microbubbles have a layer of nanoparticles, which are densely wrapped around the air core, thereby sealing the air and preventing the release of tPA loaded in the circulation. The magnetic component of the shell helps to target the microbubbles to the clot under a magnetic field. Under ultrasound stimulation, the microbubbles oscillate, and after the microflow, the shelled nanoparticles are released from the bubbles. The momentum received from the oscillation causes the nanoparticles to penetrate the agarose-fibrin gel and the femoral vein clot, respectively, reaching 1 cm and 100 microns. Permeable tPA accelerates the transfer of substances to the inside of the clot, thereby increasing the lysis rate and thrombolytic effect.
Nanoparticle shell microbubbles (MMB-SiO2-tPA) are composed of gas core and nanoparticle shell. The core is air, and the nanoparticles are a mixture of magnetic iron oxide nanoparticles (50 nm; MMB) and mesoporous silica nanoparticles (50 nm; SiO2-tPA) containing tPA. In the presence and stirring of an anionic surfactant (such as SDS), the gas is encapsulated in the surfactant, and the nanoparticles aggregate at the gas-liquid interface. Their size can be precisely controlled by adjusting the stirring speed and the concentration of nanoparticles (20).
The structure and morphology of the prepared MMB-SiO2-tPA are shown in Figure 2A. The average thickness of the nanoparticle shell is about 1.5 μm, which accounts for about 20 to 30 layers of assembled nanoparticles (Figure S1). The element mapping confirmed the presence of Fe, Si and O on the shell of MMB-SiO2-tPA, and the distribution was uniform (Figure 2B). The microbubbles are compacted into a spherical shape with an average diameter of 5.36±1.44μm (Figure 2C), similar to the microbubbles used in clinical practice (usually 2 to 8μm) (21). In order to determine whether the mesoporous silica nanoparticles were loaded in the microbubble shell, SiO2-tPA nanoparticles were replaced with SiO2-Cy5.5 nanoparticles. In the confocal image, red fluorescence surrounds the shell of most of the microbubbles, while no fluorescence is found in the hollow core, which indicates the structure of the nanoparticle shell microbubbles and the effective assembly of silica nanoparticles (Figure S2). Being out of the focal plane will cause concentrated fluorescence in the same confocal image and produce smaller-sized microbubbles. Quantitative element analysis by inductively coupled plasma emission spectrometry (ICP-OES) showed the Fe and Si content in the MMB-SiO2-tPA sample (Figure 2D), and the tPA was quantified by bicinchoninic acid (BCA) measurement Content (Figure 2E). After calculating the number of MMB-SiO2-tPA in different volumes of solution (Figure 2F), it is determined that the amounts of Fe, Si and tPA are 1.25×10-8, 2.99×10-10 and 6.63×10 per MMB-SiO2-tPA respectively -12 g. The encapsulation rate of tPA is 47.9%.
(A) Ambient scanning electron microscope image of MMB-SiO2-tPA in bright field mode. Scale bar, 10μm. (B) Scanning electron microscope and elemental map of a single MMB-SiO2-tPA. Scale bar, 5μm. (C) Diameter distribution of MMB-SiO2-tPA; n=200. (D) Quantify the iron and silicon content in different volumes of MMB-SiO2-tPA by ICP-OES. (E) The content of thrombolytic drug (tPA) in different volumes of MMB-SiO 2 -tPA determined by BCA. (F) Calculate the MMB-SiO2-tPA in different volumes of solutions. (G) In the presence of plasminogen activator inhibitor-1 (PAI-1), the relationship between in vitro enzymatic activity of natural tPA, SiO2-tPA and MMB-SiO2-tPA and time. (H) Retention activity of tPA after 3 and 12 hours in the presence of PAI-1. (I) Cumulative release curve of thrombolytic drug tPA from MMB-SiO2-tPA under different sound pressures of ultrasound. The error bars in all figures show the standard division through at least three experiments.
The circulating half-life of tPA is relatively short (approximately 2 to 6 minutes) due to the presence of inhibitors such as plasminogen activator inhibitor-1 (PAI-1; the main inhibitor of tPA) (22). When it is loaded in mesoporous silica nanoparticles, its stability can be significantly improved. For example, we compared the availability of three formulations (ie natural tPA, SiO2-tPA and MMB-SiO2-tPA) in the presence of PAI-1. When exposed to PAI-1, tPA activity in all groups decreased over time (Figure 2G). When exposed to PAI-1 for 60 minutes, the retention activity of natural tPA was reduced to 25%, while more than 50% of tPA in SiO2-tPA and MMB-SiO2-tPA kept its activity at the same time. After 12 hours of incubation with PAI-1, MMB-SiO2-tPA still maintained 36% of the tPA activity, which was higher than the 16% and 8% of SiO2-tPA and natural tPA, respectively (Figure 2H). It is worth noting that the close packing of the shelled nanoparticles on the surface of the microbubbles prevents the release of tPA from the silica nanocarrier without ultrasonic triggering, resulting in the retention activity of MMB-SiO2-tPA higher than that of SiO2 -tPA.
The microbubble-based drug delivery system is expected to release drugs after ultrasound stimulation. Traditionally, most microbubbles are stabilized by a “rigid” shell formed from polymers, silica or proteins (21, 23). The release of cargo depends on the inertial expansion and explosion of bubbles. This phenomenon is called “cavitation” and usually requires high acoustic driving force (ie, high intensity ultrasound) (24). In order to reduce the risk of tissue damage during cavitation, the release of drugs through stable microbubble oscillations activated by low-intensity ultrasound (Figure S3) is an option (20, 25, 26).
We compared the tPA release under stable oscillation and cavitation of microbubbles. When the ultrasonic intensity is set below the threshold to activate microbubble cavitation (0.4 bar or 0.04 MPa in our experiment), stable oscillation can be obtained (20). Then the shelled nanoparticles are released by the oscillation of these microbubbles accompanying the microflow. For example, when ultrasound is applied for five cycles at a sound pressure of 0.05 bar (ie 0.005 MPa; Figure 2I), about 5% of tPA is released. If you continue to apply ultrasound for another 60 cycles, the amount of tPA released will reach a stable level of 90%. Due to the stable oscillation of MMB-SiO2-tPA without microbubble collapse, the gradual release of tPA is achieved as the number of applied ultrasonic cycles increases. When most of the nanoparticles are released, MMB-SiO2-tPA gradually dissolves in the solution. By increasing the sound pressure to 0.1 and 0.15 bar, a faster release kinetics was observed compared to 0.05 bar, indicating that a stronger oscillation occurred. If the sound pressure (ie 0.5 bar or 0.05 MPa) is higher than the cavitation threshold, the bubble burst due to the cavitation effect will release nearly 90% of tPA in the first five cycles of ultrasound. Based on these findings, the on-demand release of tPA can only be achieved by stable microbubble oscillation under low-intensity ultrasound.
We first verified the response of MMB-SiO2-tPA to magnetic guidance in the extracorporeal vascular system. Dissolve MMB-SiO2-tPA in the cell culture medium and pump it into a polyethylene container (diameter 3 mm) through a syringe at a speed that simulates blood flow (1.2 cm s-1). When the magnet is placed close to the container, MMB-SiO2-tPA will accumulate on the magnet in about 2 s (Figure S4). After the magnet is removed, the accumulated MMB-SiO2-tPA quickly disperses and circulates with the liquid flow without obvious adhesion to the container wall.
The next step is to confirm this magnetically guided in vivo targeting. Ultrasound imaging can be used to track this process because microbubbles can enhance the contrast of ultrasound imaging. As shown in Figure 3B, after injecting MMB-SiO 2 -tPA into the model, the B-mode sound intensity of the model is increased by three times. The enhancement of the contrast mode is more pronounced, with a 17-fold enhancement after injection (Figure 3D).
(A) Schematic diagram of ultrasound imaging and magnetic targeting process in femoral vein thrombosis model. (B) External ultrasound phantom images of MMB-SiO2-tPA in B mode and contrast mode. (C) In vivo ultrasound images of femoral vein thrombosis before and after injection of MMB-SiO2-tPA in B mode and contrast mode. 5 minutes after the MMB-SiO2-tPA injection, place the magnet near the femoral vein. (D) The sound intensity of the region of interest (red box) quantified in the ultrasound phantom image. au, arbitrary unit. (E) The sound intensity of the region of interest (red box) quantified in the ultrasound image of the mouse model. The error bars in all figures show the standard division through at least three experiments.
An animal model was established in mice by permeating the femoral vein with ferric chloride. We imaged mice before and after intravenous injection of MMB-SiO2-tPA. Place a magnet near the thrombus for magnetic targeting (Figure 3A). Five minutes after intravenous injection of MMB-SiO2-tPA, the sound intensity of B mode and contrast mode of thrombus in femoral vein increased slightly, which may be caused by the presence of a small amount of MMB-SiO2-tPA at room temperature. Thrombosis is formed through blood circulation (Figure 3C). After applying the magnet, it was observed that the sound intensity of the B mode and the contrast mode were significantly increased (1.4 times and 2.6 times, respectively), indicating that MMB-SiO2-tPA was accumulated at the thrombus site through magnetic targeting (Figure 3E). Therefore, MMB-SiO2-tPA can be magnetically guided to the thrombus and used as an ultrasound contrast agent for thrombus diagnosis, while non-invasively monitoring the delivery efficiency of the therapeutic agent.
During fibrinolysis, tPA activates plasminogen and converts it into plasmin, which can cleave fibrin into soluble products (27). In order to verify the effectiveness of fibrinolysis and the penetration of tPA in three dimensions, a vertical channel gel system composed of fibrinogen, thrombin, plasminogen and agarose was developed. The fibrin formed by the reaction of fibrinogen and thrombin is dispersed in the agarose gel with good uniformity and is pale white throughout the gel (Figure 4A, upper left). Once fibrin interacts with tPA, the degradation products will make the gel transparent (dark areas), indicating areas of tPA cleavage. As shown in Figure 4, after adding tPA from the top of the vertical channel, starting from the top of the gel, the lysis area gradually increased over time (Figure 4A, bottom left). The gradual dissolution of tPA is a standard clinical thrombolysis process, due to the mass transfer kinetics of tPA at the liquid-gel (or liquid-thrombus) interface, indicating that the penetration of tPA in the gel is limited. Similar findings were also found in the group treated with SiO2-tPA (Figure 4A, upper right). Due to the delayed release of natural tPA from the SiO2 nanocarrier, slower lysis was observed compared with natural tPA (Figure S5). ). In order to promote the penetration of SiO2-tPA into the gel, MMB-SiO2-tPA (equivalent to the amount of tPA and natural tPA or SiO2-tPA groups) is concentrated at the liquid-gel interface of the magnet, and then washed by low pressure. Intensity ultrasound (0.2 bar, or 0.02 MPa) to stabilize the oscillation. Once stable oscillation occurs, the penetration of iron oxide and silica nanoparticles through the entire gel will be immediately observed, with black dots going deep into the gel along the vertical channel (Figure 4A, bottom right). The expansion of the dark zone at multiple locations indicates that cleavage occurs not only at the liquid-gel interface, but also around the nanocarrier that penetrates into the gel. Therefore, after 6 hours of treatment, the fibrinolytic efficacy of MMB-SiO2-tPA (quantified by the fibrinolytic zone; Figure 4B) is higher than that of natural tPA. Within 6 to 12 hours after treatment, the average fibrinolysis rate obtained by MMB-SiO2-tPA increased significantly (Figure 4C). However, due to the consumption or degradation of tPA, the fibrinolysis rate obtained by natural tPA decreases with time. It is worth noting that compared with natural tPA, these improvements of MMB-SiO2-tPA on fibrinolysis efficiency and fibrinolysis rate are realized in a closed space without blood flow. Considering the blood flow in the body that leads to the rapid clearance of tPA, the improvement of tPA permeability will keep the delivered tPA in the blood clot, and finally accelerate the thrombolytic effect and improve the efficacy of tPA treatment.
(A) Schematic diagram and representative photos of the fibrinolysis process of agarose fibrin gel incubated with saline, natural tPA, SiO2-tPA and MMB-SiO2-tPA under different thrombolysis times. (B) Quantification of the fibrinolytic area of ​​fibrin incubated with saline, natural tPA, SiO 2 -tPA and MMB-SiO 2 -tPA over time (n = 5; *** P <0.001). (C) The average fibrin dissolution rate of fibrin at different time intervals incubated with natural tPA, SiO2-tPA and MMB-SiO2-tPA (n = 5).
The thrombolytic effect was further evaluated in isolated blood clots prepared from fresh mouse blood and thrombin (Figure 5A). As it dissolves, the clot in the tube shrinks and the supernatant turns red. As expected, the clot treated with brine hardly dissolved within 12 hours, indicating the stability of the prepared clot. When the blood clots are treated with natural tPA, SiO2-tPA or MMB-SiO2-tPA, all blood clots are gradually broken down into smaller sizes, and the supernatant gradually changes from colorless to blood red (Figure 5B). The black appearance in the MMB-SiO2-tPA treatment group was caused by the released iron oxide nanoparticles. The efficiency of lysis was quantified by measuring the mass loss of blood clots at 3 and 12 hours after treatment. In the first 3 hours (Figure 5C), the blood clot treated with MMB-SiO2-tPA showed a hemolysis efficiency similar to that of natural tPA (about 32%) (about 27%), higher than that of SiO2-tPA or saline treatment (17 and 7% respectively). After 12 hours (Figure 5D), compared with the dissolution efficiency (68%, 51% and 16%) obtained by natural tPA, SiO2-tPA or brine treatment, MMB-SiO2-tPA treatment reached the highest dissolution efficiency. About 93%. , Respectively). The amount of hemoglobin in the supernatant (released during clot dissolution) 12 hours after treatment showed a trend similar to the dissolution efficiency, confirming the results obtained in the in vitro experiment. Since this experiment was performed in a static state without blood flow, the improved dissolution efficiency of the MMB-SiO2-tPA treatment group may be attributed to the enhanced permeability and retained activity of tPA.
(A) Schematic diagram of the process of dissolving blood clot by magnetic field combined with low-intensity ultrasound. (B) Representative images of the thrombolysis process at 0, 3, 6, 9 and 12 hours after treatment with salt water, natural tPA, SiO2-tPA and MMB-SiO2-tPA, respectively. (C) Quantify the dissolution efficiency by measuring the mass loss of the blood clot 3 hours after treatment. (D) Quantify the dissolution efficiency by measuring the mass loss of the blood clot 12 hours after treatment. (E) The absorbance value of the supernatant 12 hours after treatment (λ=540nm); n=5; *P<0.05, **P<0.01 and ***P<0.001.
In order to study whether the blood clot penetration strategy is applicable to in vivo animal models, we performed different treatments on femoral vein thrombosis in mouse models. Male C57/BL6J mice were pretreated with FeCl3 to infiltrate and form blood clots, and then MMB-SiO2-tPA was injected through the tail vein, followed by magnetic targeting and low-intensity ultrasound treatment (Figure 6A). The control group included mice injected with saline, natural tPA and SiO2-tPA, respectively. Images of thrombus were recorded every three hours, and then the mice were sacrificed 12 hours after treatment. As shown in Figure 6B, the femoral vein showed a dark area where the FeCl 3 filter paper was placed, indicating that a blood clot was successfully formed. Over time, the dark areas of the femoral vein treated with saline became longer, indicating the progression of venous thrombosis. When natural tPA is injected intravenously, the size of the dark area shrinks and becomes lighter, indicating that the dissolution of the blood clot gradually occurs over time. With this natural tPA dose (100μl, 10μgml-1), the blocked vein cannot be completely recanalized, because light shadows will still appear 3 and 12 hours after treatment. Although SiO2-tPA has the ability to protect natural tPA from inhibitors in the blood, SiO2-tPA treatment only achieves a thrombolytic effect similar to that of natural tPA treatment, which indicates that SiO2-tPA has a lower delivery efficiency for blood clots. In the case of the same amount of tPA, the dark area of ​​the vein treated with MMB-SiO2-tPA disappeared completely even 3 hours after treatment. The histological results of the femoral vein (Figure 6C) are in good agreement with the results in Figure 6B. Ideally, the thrombus is considered a cylinder, and the quantification of the clot volume reveals the thrombolytic effect. Or, by measuring the cross-sectional area of ​​blood clots and veins in histological images to quantify the area of ​​thrombus (percentage of vein lumen), the area of ​​thrombus formation can be roughly estimated (28). However, MMB-SiO2-tPA treatment achieves the best thrombolytic effect, with the smallest thrombus area (about 13%), while the percentages of treatment with saline, natural tPA or SiO2-tPA are 66%, 40% and 50%, respectively. Respectively (Figure 6D). It is worth noting that, according to histological analysis (Figure 6C and Figure S7), two of the four femoral veins treated with MMB-SiO2-tPA achieved complete recanalization. In addition, the histological image of the clot proved that the permeability of the released nanoparticles was improved (Figure 6E). The shelled nanoparticles (black dots) are mainly attached to the periphery of the blood clot without ultrasound triggering. When triggered by low-intensity ultrasound, the shelled nanoparticles are released and penetrate into the blood clot, resulting in a relatively uniform distribution of the nanoparticles in the blood clot.
(A) Schematic diagram of the treatment procedure of a mouse model of femoral vein thrombosis. (B) Representative images of thrombolytic evaluation after treatment with brine, natural tPA, SiO2-tPA and MMB-SiO2-tPA. The white arrow indicates the induced thrombus (n = 4). (C) Representative histological analysis of femoral vein after 12 hours of treatment with saline, natural tPA, SiO2-tPA and MMB-SiO2-tPA (n = 4). Scale bar, 50μm. (D) Quantification of thrombus area (venous lumen%) in femoral vein in different treatment groups (n=4). (E) Representative histological analysis and magnetic targeting of femoral vein after MMB-SiO2-tPA with or without low-intensity ultrasound treatment. The red arrow indicates the enrichment of MMB-SiO2-tPA, and the red circle indicates MMB-SiO2-tPA with complete structure. Scale bar, 50μm.
In order to evaluate the safety of the precise delivery strategy in vivo, mice were injected with MMB-SiO2-tPA intravenously, and the main organs were collected. As shown in the histological image (Figure S8A), there was no obvious organ damage or inflammatory lesions in the short-term (ie 1 day) and long-term (ie 7 days). At the same time, by comparing healthy mice and mice injected with MMB-SiO2-tPA (7 days later), serum biochemical analysis and whole blood plate detection were studied (Figure S9). The results showed that there was no significant difference in all measurement indicators between the two groups, indicating that MMB-SiO2-tPA has good biocompatibility. After 24 hours of injection, SiO2 nanoparticles mainly aggregated in the liver and kidney, while iron oxide nanoparticles were mainly distributed in the spleen (Figure S10). The biodistribution of MMB-SiO2-tPA in mice indicates that they are cleared by the reticuloendothelial system.
In addition, due to off-target effects, thrombolytic drugs always cause bleeding complications. For example, circulating tPA breaks down fibrinogen in the blood, leading to abnormal hemostasis and bleeding side effects (29). In order to evaluate the effect of MMB-SiO2-tPA on hemostasis, the tail bleeding time was tested on a mouse model. First, mice were injected intravenously with saline, natural tPA or MMB-SiO2-tPA (with the same amount of tPA), and then their tails were cut with a scalpel (Figure S8B). The tail bleeding time (approximately 7.6 minutes) of the mice treated with natural tPA was almost four times that of the saline treatment group (approximately 1.8 minutes), indicating obvious abnormal hemostatic side effects. In contrast, the bleeding time of MMB-SiO2-tPA administration without ultrasound trigger was similar to that of the saline group (approximately 2.2 minutes), indicating that the off-target effect was limited and bleeding complications were frequent.
Last but not least, the safety assessment of ultrasound intensity on vascular damage in mice was carried out. High-intensity ultrasound (above 0.04 MPa, or 0.4 bar) used to activate air pockets may cause endothelial damage and cerebral hemorrhage (in stroke treatment), which may cause safety issues (30-32). MMB-SiO2-tPA was injected intravenously into mice and targeted to the tail vein, and then subjected to ultrasound treatment, the intensity of which triggered bubble cavitation (0.5 bar) and stable oscillation (0.2 bar), respectively. as the picture shows. In S8C, due to bubble cavitation, deformation of the blood vessel wall treated by high-intensity ultrasound was observed. The blood vessel wall becomes wrinkled, thinned or even ruptured at the thinnest point (black arrow in Figure S8C). On the contrary, when the ultrasound intensity is reduced by more than half, the blood vessel wall remains intact and the structure does not deform. Therefore, the precise delivery strategy of low-intensity ultrasound shows satisfactory safety, with limited bleeding complications and vascular damage.
This work proposes a precise delivery strategy to achieve targeted and controlled delivery of thrombolytic drugs. In this process, three conditions must be met, namely the maintenance of the activity in the circulation, the penetration of the clot and the penetration of the clot.
In order to keep the activity of the drug in the circulation, we first load tPA into mesoporous silica nanoparticles to protect the activity of tPA from the influence of tPA inhibitors in the blood. In addition, the stability of nanomedicine in the circulation is also important for the maintenance of tPA activity and bioavailability. In our design, nanoparticles with different functions are integrated into ultrasound-responsive microbubbles through self-assembly and stabilized through the “buckling effect”. Specifically, when a strong shear flow is generated during the stirring of the mixed solution, microbubbles are first formed due to air entrainment and breaking. Due to the hydrophobic surface properties, the nanoparticles self-assemble at the microbubble interface. When the resulting solution is stored in the environment, the microbubbles shrink due to gas diffusion, while the nanoparticles remain attached to the interface. The nanoparticles will not bend until the bubble size is reduced to small enough for tight packing (Figure 1B). On the one hand, this curvature prevents the reduction of the bubble surface area; on the other hand, this curvature prevents the reduction of the bubble surface area. On the other hand, the multilayer packing also shields the gas from diffusing out of the gas core. In the end, these microbubbles reached a balanced size with a multilayer nanoparticle shell, thereby achieving excellent stability. The close packing of the nanoparticles also prevents the loaded tPA from being released into the blood, thereby reducing the possibility of contact between tPA and its inhibitor. Compared with the delivery strategy that uses tPA surface binding or uncontrolled release of tPA, this strategy can improve the delivery efficiency of active tPA to blood clot (12, 19). In the presence of its inhibitors, the activity of tPA can maintain a half-life of 1 hour (Figure 2H), which is much longer than the activity of natural tPA (about 5 minutes). The stability of MMB-SiO2-tPA in circulation was confirmed by US imaging in a mouse model, and contrast enhancement was observed in blood vessels even after 30 minutes after injection. Therefore, the precise delivery strategy meets the first criterion in the thrombolytic process of nanomedicine, which is to maintain the activity of tPA in the blood circulation.
Targeted delivery improves delivery efficiency and specificity, thus reducing the required dosage and side effects. Due to the protein corona and relatively low targeting efficiency of nanomedicine formed in blood, use RGD (Arg-Gly-Asp) motif (which binds to active platelet integrin GPIIb/IIIa) or anti-fibrin antibody target This strategy is still challenged by relatively low targeting efficiency. The heterogeneity of individual blood clots (33-35). For example, there was no significant difference in the recanalization rate of rabbit aortic thrombosis by injecting tPA-loaded echogenic liposomes with or without sonication (15). Ultrasound is believed to enhance the catalytic activity of exposed liposome-associated tPA, rather than causing the release of enzymes into the blood clot. The increase in thrombolytic efficacy may be impaired by poor delivery efficiency (no targeting strategy) and limited penetration of tPA in blood clots. Using porous magnetic microrods as a carrier, a method of magnetically targeted delivery of tPA to the blood clot of the mouse middle cerebral artery has been achieved in the reported work (19). In our strategy, magnetic targeting technology can be simply applied to adjacent tissues of thrombus diagnosed by ultrasound imaging (Figure 3E), so that it can meet the second criterion, and it is promising and clinically feasible .
Due to the structure of the thrombus, which is composed of abundant platelets and well-organized fibrin, the thrombus penetration strategy is reported to be limited. The rotating magnetic microrod may generate mechanical force on the cross-linked fibrin, thereby destroying the structure of the blood clot and promoting the penetration of the released tPA (19). The micro-stick containing tPA [tPA (0.13 mg kg-1)] restored the blood flow in a mouse model of cerebral artery occlusion within 25 minutes, which is higher than only at high concentrations [tPA (10 mg kg-1)]. The significant increase in lysis rate and the greatly reduced dosage of tPA are partly attributable to the increase in the release rate of tPA and the improvement in mass transportation, and partly to the destruction of the fibrin network by mechanical rotation force, which leads to the permeability of tPA. Improve (19). In the current work, we aim to show direct evidence of the enhanced penetration of tPA nanocarriers in clot tissue by low-intensity ultrasound. Previously, we have demonstrated that the tissue permeability of nanoparticles is improved by stable microbubble oscillation (20). In short, self-assembled nanoparticles form an “elastic shell” due to the weak hydrophobic interaction between the nanoparticles. When the ultrasonic resonance intensity is lower than the cavitation threshold, the microbubbles are activated by their resonance frequency to make them oscillate stably. as the picture shows. S3, under high sound pressure, the size of the microbubbles is reduced, and the shelled nanoparticles are tightly packed. At low sound pressure, the microbubbles expand, and the shelled nanoparticles are loosely packed, accompanied by the nanoparticles falling off in the outermost layer. Subsequently, when the sound pressure increases again, the loosely packed nanoparticles reassemble to form a curved shell. This reassembly of the shelled nanoparticles was recorded in a supplementary film by a charge-coupled device camera. The movement of the fluorescent silica nanoparticles in the shell reveals the reassembly process of the nanoparticles during the stable microbubble oscillation process. It is worth noting that the release and movement of silica nanoparticles along with the micro-flow caused by stable oscillations suggests the possibility of improved tissue permeability. The released nanoparticles can penetrate up to 1 cm in the agarose-fibrin gel (Figure 4A). According to the literature (13), the cleavage process can be described as S + T→KTST(SP)→KPS + P, where S, T, ST, SP and P are the exposed sites of lysine, tPA molecule, tPA-lysine Acid complex, tPA-product complex and product KT and KP are the tPA absorption rate and product desorption rate, respectively. Assuming that CtPA = 10μgml-1 (KT <
In the mouse model, the released nanoparticles can easily reach the center of the venous thrombosis with a diameter of 300 μm (Figure 6E). Therefore, lysis occurs not only at the interface of the clot, but also at many internal locations, leading to accelerated thrombolysis. In the mouse venous thrombosis model, compared with the traditional tPA injection [tPA (0.03 mg kg-1)], when treated with MMB-SiO2-tPA [tPA (0.03 mg kg-1)], the residual thrombus was reduced 67.5%. Note that such a low tPA dose of MMB-SiO2-tPA can achieve complete recanalization (two of the four femoral veins). By increasing the dose of tPA loaded for time-critical thrombolytic therapy, the therapeutic effect and lysis rate can be further improved. The increase in tPA permeability finally meets the third criterion of the thrombolytic process.
Compared with femoral vein thrombosis, the challenge of delivering thrombolytic drugs to complex sites such as cerebral embolism, pulmonary embolism and myocardial infarction is greater. Although encouraging results have been shown both in vitro and in vivo, the delivery strategy of the present invention can be further improved for embolization in complex locations. First, the size of MMB-SiO2-tPA can be reduced to avoid rapid removal of the reticuloendothelial system. Finally, due to its hydrophobic surface properties, the cycle time of MMB-SiO2-tPA has been challenged. Cell membrane (such as red blood cells and platelets) coating technology can be used to extend the circulation of MMB-SiO2-tPA, thereby improving delivery efficiency and therapeutic effects (36-39).
All in all, we concluded that our precise delivery strategy can complete the three important steps of nanomedicine for thrombolysis, namely maintaining activity in the circulation, targeting the blood clot and penetrating the blood clot. The increase in hemolysis rate and therapeutic efficacy is attributed to the increase in the effective concentration of tPA at the clot site, which is the result of the maintenance of tPA activity, magnetic targeting and the increase in the permeability of tPA in the clot. Therefore, this strategy has broad prospects in thrombosis diagnosis and accelerated thrombolysis while reducing the risk of tPA complications and high-intensity ultrasound damage to blood vessels.
tPA was purchased from Merck (USA). SDS, thrombin from human plasma, plasminogen from human plasma, and protease substrate Hd-isoleucyl-1-prolyl-1-arginine-p-nitroaniline (S-2288) From Sigma-Aldrich (USA). Mesoporous silica nanoparticles (SiO2) were purchased from Shanghai So-Fe Biomedical (China). Fe3O4 nanoparticles were purchased from Alfa Aesar (USA). Fibrinogen from human plasma was purchased from Shanghai Yuanyu Biotechnology Co., Ltd. (China). Agarose was purchased from BD (USA). All other chemicals and solvents were purchased from Sigma-Aldrich.
The microbubbles with nanoparticles were prepared by the previous method (20). In short, the magnetic nanoparticles (Fe3O4) are dispersed in deionized water to form a stock solution (10 mg ml-1), and ultrasonicated for 20 minutes before use. Next, homogenize the mixed solution containing 150μl SiO2-tPA nanoparticles (0.2 mg ml-1), 150μl SDS (10 mM-1) and 400μl Fe3O4 nanoparticles (10 mg ml-1) at 20,000 rpm for 3 minutes. The microbubbles with nanoparticles were stabilized overnight to tightly pack the nanoparticles, and then purified by magnetic separation three times with deionized water.
Observe the morphology and size with a microscope (Olympus IX71, Japan) and an environmental scanning electron microscope (Philips XL30, Netherlands). Manually measure the diameter from the photo and count at least 200 microbubbles. A laser scanning confocal microscope (Olympus FV1000MPE, Japan) was used to image the fluorescent nanoparticle shell microbubbles. The content of iron and silicon in different volumes of MMB-SiO2-tPA was measured by ICP-OES (PerkinElmer, USA). The content of tPA in MMB-SiO2-tPA was tested by BCA protein determination kit.
As previously reported (40), the chromogenic substrate S-2288 was used to test the fibrinolytic activity of tPA. At 37°C, natural tPA was added to a microtiter plate containing assay buffer [0.1 M-1 tris-HCl (pH 7.4)] and S-2288 (1.0 mM-1). Fibrinolytic activity was calculated by ΔAbs/min at 405 nm in a 30-minute reaction. The inhibitory efficiency of tPA was determined by incubating the same amount of tPA (10 μgml-1) and active PAI-1 (0.5 nM-1) in 200 μl assay buffer in a microtiter plate at 37°C for a predetermined time. Then, the residual activity of tPA was measured by the above method.
Different intensities of ultrasound were tested in vitro to release tPA from MMB-SiO2-tPA. In short, the ultrasonic frequency from 10 to 900 kHz is adjusted by a function generator (Keysight, USA), the amplitude is from 2 to 20 Vpp (peak-to-peak voltage), and the ultrasonic power is adjusted from 0.1% to 10%. Amplifier (American T&C). Ultrasound is applied through a self-made focusing transducer, and each cycle contains a duration of 5 s with an interval of 1 s. The output ultrasonic intensity at the focus of the transducer was monitored by an oscilloscope (Keysight, USA). After different ultrasonic cycles, the supernatant was collected, and the amount of tPA released was quantified by the BCA protein determination kit.
A gel mold with holes was used as an ultrasonic phantom, and 1 ml of MMB-SiO2-tPA solution was added. A high-resolution micro-imaging system (VisualSonics Vevo 2100, Canada) was used to image the model in 18 MHz static and static modes (using both B mode and contrast mode) using a transducer. The center frequency, intensity power and contrast gain are set to 18 MHz, 10% and 35 dB, respectively. The average video intensity in the region of interest (ROI) was analyzed by ultrasound imaging software.
For in vivo ultrasound imaging, the femoral vein of male C57/BL6J mice was treated with 20% ferric chloride solution. Then, the mice were anesthetized with a 10% chloral hydrate solution and imaged by an ultrasound imaging system. After 5 minutes of intravenous injection of 100 μl MMB-SiO2-tPA, the femoral vein was imaged again, and then a magnet was placed. The accumulation of MMB-SiO2-tPA by the magnet was monitored over time by ultrasonic imaging. The average video intensity in the ROI was analyzed by ultrasound imaging software.
Combine 10 ml agarose solution (0.5%) containing 20 μl thrombin solution (250 U ml-1) with 1 ml fibrinogen solution (10 mg ml-1) and 10 μl plasminogen solution (1 mg ml-1) ) Mix thoroughly). Subsequently, the mixed solution was uniformly added to the vertical channel and incubated at 37°C for 2 hours to form a fibrin gel. The concentration of natural tPA, SiO2-tPA and MMB-SiO2-tPA used in in vitro, in vitro and in vivo experiments is fixed with the same amount (1μg) of tPA (natural tPA, 100μl of 10μg) ml-1; SiO2-tPA, 100μl 50μgml-1; MMB-SiO2-tPA, 100μl concentrated MMB-SiO2-tPA, the quantity is 1.5×106 ml-1). Then, add 100μl of normal saline, natural tPA, SiO2-tPA or MMB-SiO2-tPA and equal concentration of tPA (10μgml-1) to the channel on the top of the gel, and incubate at 37°C for 3 and 6 respectively , 9 and 12 hours. For the MMB-SiO2-tPA treatment group, place a magnet on the bottom of the channel and apply ultrasound at 0.2 bar intensity for 3 minutes. Finally, the fibrinolytic activity of each sample was evaluated by comparing the dark areas of the gel. Briefly, the image is first processed to remove the background and then converted into a binary (black and white) image. The “black” pixel area represents the lysis area, and the “white” pixel area represents the agarose fibrin gel. In order to identify the boundary, the threshold is determined by processing the images of the tPA group. The threshold has been adjusted to include all black areas within the selected area threshold. Then, the same threshold is applied to the processing of all other images.
The blood clot was prepared by the previous protocol (41). C57/BL6J (8 to 10 weeks old) male mice were anesthetized by isofluoride gas. One hundred microliters of fresh blood was obtained from the orbital vein and distributed into several centrifuge tubes containing 50 U of thrombin solution. Place the test tube at 37°C for 3 hours and then move to 4°C for 3 days.
Put the prepared blood clot into a centrifuge tube containing 1 ml of saline. Then, physiological saline (100μl), natural tPA, SiO2-tPA, and MMB-SiO2-tPA with the same concentration of tPA (10μgml-1) were added to the solution and incubated at 37°C. During the incubation, the lysis process is monitored at predetermined time points. For the MMB-SiO2-tPA treatment group, a magnet was placed under the blood clot and ultrasound was applied at 0.2 bar intensity for 5 minutes. Record the weight of the blood clot during the lysis process and calculate the efficiency of blood clot lysis. In addition, the supernatant of all samples after 12 hours was collected and measured (light absorption) at OD 540 (optical density of 540).
All procedures involving animals were approved by the Institutional Animal Care and Utilization Committee of Nanyang Technological University. Male C57/BL6J mice (6 to 8 weeks old) were obtained from Nanjing Qinglongshan Animal Breeding Farm. Induce femoral vein thrombosis according to the previous protocol. Briefly, mice were anesthetized with 10% chloral hydrate (100 μl) by intraperitoneal injection. Expose the left femoral vein of the mouse with a scalpel and forceps. After exposure, the filter paper soaked with 20% ferric chloride solution was placed on the surface of the femoral vein for 1-2 minutes. Then, the filter paper was removed, and the container was washed with sterilized phosphate buffered saline. Finally, visible femoral vein thrombosis is formed.
In order to study the efficacy of thrombolysis in vivo, 100μl of normal saline, natural tPA, SiO2-tPA and MMB-SiO2-tPA and the same amount of tPA (10μgml-1) were injected intravenously (n=4 per group). For the MMB-SiO2-tPA treatment group, a magnet was placed near the blood clot for 25 minutes, and then ultrasound was applied at 0.2 bar for 5 minutes. Monitor the thrombolysis process by taking pictures for the next 12 hours. After euthanasia, vascular tissue was excised from the mice and collected for histological analysis (n = 4 per group). Unknowing researchers used Image J software to process and analyze the slices. The area of ​​the blood clot is measured, and the thrombolytic efficiency is determined by the ratio of the area of ​​the blood vessel obstruction to the total vascular system.
In order to evaluate the improvement of nanoparticle penetration in vivo, mice with femoral vein thrombosis were divided into two groups (n=3 in each group). One hundred microliters of MMB-SiO 2 -tPA was administered by intravenous injection, and the magnet was placed near the thrombus for 25 minutes. Subsequently, the two groups received or did not receive low-intensity ultrasound (0.2 bar) treatment. After sacrifice, vascular tissue was excised from the mouse and collected for histological analysis.
Inject 100μl MMB-SiO2-tPA intravenously into male C57/BL6J mice (6 to 8 weeks old). Then, 1 or 7 days later, the mice were euthanized, and the main organs were collected for histological analysis. Healthy mice injected with saline intravenously were selected as the control group (n = 3 in each group).
Tail bleeding analysis was performed by the previous method (29). Male C57/BL6J mice were anesthetized with 10% chloral hydrate by intraperitoneal injection. Subsequently, 100 μl saline, natural tPA and MMB-SiO2-tPA were administered with the same concentration of tPA (10 μgml-1) (n=3 per group). After 5 minutes, use a scalpel to remove the 1 cm distal tail from the mouse. Record the hemostasis time (bleeding stops completely at least 1 minute).
In order to study ultrasound damage to blood vessels, male C57/BL6J mice were anesthetized with 10% chloral hydrate by intraperitoneal injection. Thereafter, 100 μl of MMB-SiO2-tPA was administered at an equivalent concentration of tPA (10 μgml-1). Then, place the magnet in the middle of the mouse’s tail. Apply ultrasound of different intensity (0.2 and 0.5 bar) respectively. Finally, a 5 cm distal tail was removed from the mouse for histological analysis.
All data are expressed as mean ± SD. Use SPSS software to compare and analyze between and within groups in each experiment through unpaired Student’s t test and one-way analysis of variance (ANOVA). A probability (P) value of <0.05 is considered statistically significant.
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Wang Siyu, Guo Xixi, Xiuweijun, Liu Yang, Ren Lili, Xiao Huaxin, Yang Fang, Yu Yu, Xu Chenjie, Wang Lianhui
Targeted and clot-penetrating dissolution of thrombolytic drugs by microbubble oscillation can accelerate thrombolysis.
Wang Siyu, Guo Xixi, Xiuweijun, Liu Yang, Ren Lili, Xiao Huaxin, Yang Fang, Yu Yu, Xu Chenjie, Wang Lianhui
Targeted and clot-penetrating dissolution of thrombolytic drugs by microbubble oscillation can accelerate thrombolysis.
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Post time: Dec-01-2020