Hepatic fibrosis is associated with hepatocytes damage, leading to an abnormal increase in blood ammonia. Accumulating ammonia further aggravates the pathological environment and can trigger hepatic encephalopathy. Therefore, it is meaningful to construct therapeutic platform for targeted ammonia clearance. In this work, a biocompatible water powered Zn micromotor is constructed as ammonia chemotaxis platform, which can be actuated by water splitting reaction and the self-generated Zn2+ gradient. It can propel towards NH3·H2O source through the formation of complex ions [Zn(NH3)1](OH)+ and [Zn(NH3)2](OH)+, representing a generalizable chemotaxis strategy via coordination reaction. In vivo, the biomimetic swarming behavior of Zn micromotors allows precise navigation and reduction of the intrahepatic ammonia level, thus reshaping the local pathological environment. The decrease in blood ammonia leads to a reduction in ammonia permeated to the brain, which in turn alleviates neuron damage and inhibits the development of hepatic encephalopathy. This mechanism, operating in a green, zero-waste manner, facilitates integration of these micromotors into domain of biological regulation. Such environment adaptive platform is favorable for targeted treatment of hepatic fibrosis and hepatic encephalopathy caused by hyperammonemia, which is expected to provide inspiration for future personalized and precision medicine.
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Chemotactic Zn micromotor for treatment of high blood ammonia associated hepatic encephalopathy
https://doi.org/10.21203/rs.3.rs-4933091/v1
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chemotaxis
micromotors
hepatic encephalopathy
coordination
targeting therapy
Ammonia is a natural endogenous molecule1. In vivo, blood ammonia primarily originates from the catabolism of amino acids and proteins by bacteria in the gut, hydrolysis of urea and deamidation of amino acids in liver2. The produced ammonia is typically metabolized in liver through catalysis of low-toxic glutamine from NH4+ by glutamine synthetase3 or ammonia conversion into urea4–6. Under normal conditions, the liver maintains blood ammonia at a low level of dynamic equilibrium. However, upon metabolic function of hepatocytes decreases, excessive accumulation of ammonia in the liver is resulted, along with a rise in blood ammonia levels7. Ammonia accumulation results in inflammatory necrosis of liver cells, even liver fibrosis and cirrhosis8,9. Hyperammonemia can further lead to hepatic encephalopathy10–12,13, with clinical manifestations include behavioral disorders, and even coma14,15. Ammonia cross the blood-brain barrier as NH3 by free diffusion, and to a lesser extent as NH4+ via potassium ion carriers16–18. Astrocytes are the only brain cells capable of metabolizing ammonia; however, excessive ammonia causes astrocyte edema19–22, which inhibit the possibility of neuronal repair, causing irreversible brain damage23,24. Therefore, targeted ammonia clearance is meaningful for the hepatic encephalopathy therapy.
In nature, a wide variety of chemical attractants exist25. These chemical gradient can attract or repel corresponding organisms. In the macroscopic world, insects locate food by chemotaxis to food and are attracted to pheromones released by the opposite sex for mating26. Similarly, inflamed tissues release specific chemicals (chemokines) that guide neutrophils along the concentration gradient27–29. Inspired by the chemotactic behavior of organisms in nature, researchers engineered artificial micro/nanomotors capable of chemotactic movement mimicking microscopic organisms30–32. These synthetic micro/nanomotors autonomously move by converting chemical33,34, acoustic35, optical36, electrical37 and magnetic38 energy into mechanical energy, and follow the environment cues towards areas of higher chemical fuel concentration39,40, which holds promise for precision medicine41.
Chemotaxis can arise from thermodynamic driving force that lowers chemical potential with favorable substrate binding, yet systems beyond enzyme-substrate have few been described. Here, we proposed a water driven Zn micromotor (Fig. 1a) via the reaction of Zn with H+ in water. The Zn micromotors can demonstrate chemotactic migration towards NH3·H2O (Fig. 1b) via the coordination between Zn and NH3·H2O. The concentration gradient generated by the diffusion of ammonia promoted the swarming chemotaxis of Zn micromotors. We presented for the first time a kinetic system capable of autonomous chemotaxis through coordination. For hepatic encephalopathy caused by excessive blood ammonia resulting from liver injury, the central problem was to reduce the concentration of ammonia in the body. The amine absorption into blood occur in the colon and the main metabolizing organ is liver, both of which are highly concentrated sites of blood ammonia6. The chemotactic migration of Zn micromotors towards ammonia could be directed to the colon and liver. Upon arrival, local ammonia was trapped through coordination reaction, which promoted local ammonia metabolism, and improved cirrhosis/hepatic encephalopathy (Fig. 1c). This autonomous chemotaxis platform provides a new direction for the field of precision medicine.
Morphology characterization and motion mechanism of Zn micromotors
The template approach was utilized to prepare the Zn micromotors (Fig. 1a), with a polycarbonate membrane (average pore size of about 400 nm) to control the size and morphology of the micromotors. Initially, a gold layer approximately 100 nm thick was sputtered on one side of the polycarbonate film to enhance the conductivity of the template. Then, the gold-plated PC film was used as the working electrode to deposit Zn (three electrode method) to form a tubular structure. Organic reagent was then employed to dissolve the PC film, and the harvested Zn micromotors were dispersed in deionized water (DI) (Fig. 2a). Following the preparation, the morphology of the Zn micromotors were characterized by scanning electron microscopy (SEM) (Supplementary Fig. 1). The length and diameter of the Zn micromotors were determined using ImageJ software, with a length of 3.07 ± 0.65 µm (Fig. 2b) and a diameter of 0.41 ± 0.08 µm (Fig. 2c). The quantitative distribution of Zn elements was analyzed using energy dispersive X-ray spectroscopy (EDX) (Supplementary Fig. 2). As shown in Fig. 2d and 2e, the wall of the Zn micromotor was thicker at one end (bottom) and thinner at the other end (top) due to the gradual deposition from bottom to top inside the template by controlling the deposition timing. The distribution of Zn further showed that there is a Zinc content difference between the two ends. A part of the electrodeposited PC film was captured, and it was observed by SEM that hollow tubular structure was formed along the template wall (Supplementary Fig. 3). Horizontal surface of Zn micromotors was enlarged (Fig. 2f), and the distribution of Zn elements was analyzed by EDX (Fig. 2g, Supplementary Fig. 4). As shown in Fig. 2h, the Zn content was quantitatively analyzed (Zinc content 5.29%) and longitudinal planes (Zinc content 8.25%).
Zn micromotors were harvested from the template and dispersed in water. The micromotors were observed through a microscope to investigate the mechanism of motion42. It was found that the Zn micromotors performed self-propelled motion in water in a circular manner. Its autonomous motion was primarily due to the reaction between Zn and H+ in water, with the reaction formula 
(Fig. 2a). Since Zn was gradually deposited along the membrane pores from the bottom of the PC film template (near the Au end) upwards during the fabrication by the electrochemical deposition method, the Zn micromotor was of an asymmetric structure, being thicker at one end (near the gold layer end) and thinner at the other end (far from the gold layer end). As the reaction progressed, the Zn2+ accumulates higher than that at the thicker end of the micromotor wall. Therefore, it created a Zn2+ concentration gradient around the Zn micromotor, resulting in a self-generated electric field. The direction of the electric field was from the thin end to the thick end, driving the Zn micromotor towards the higher potential (thinner micromotor wall). The motion trajectories of the Zn micromotors over time were plotted as shown in Fig. 2i, Supplementary Fig. 5 and Supplementary Movie 1. The average speed (1.78 ± 1.02 µm/s) and directivity (0.19) of the micromotors were calculated (Fig. 2j, Supplementary Table 1). The spiral forward motion was the typical moving pattern of Zn micromotors. No bubbles were produced during the movement. The moving mechanism of the Zn micromotor was further demonstrated with a Zn2+ fluorescent probe (Zinquin), which combines with Zn2+ to produce fluorescent signal (Supplementary Fig. 6). As shown in the Fig. 2k, there was fluorescence aggregation around the Zn micromotor, and the fluorescence intensity was positively correlated with the concentration of Zn2+. The fluorescence intensity showed a gradually decreasing trend from one end of the Zn micromotor to the other end, which proved that there was indeed a Zn2+ concentration gradient field around the micromotors.
Collective chemotaxis dynamics of Zn micromotors
The movement mechanism of single Zn micromotor has already been demonstrated. While previous micromotors can move autonomously, they often exhibit poor directionality and lack the capability to perform complex tasks. However, it has been observed that cells and microorganisms in nature can respond to chemical attractants, leading to collective chemotactic behavior. Inspired by this natural phenomenon, we designed a biomimetic chemotaxis system, Zn micromotors for collective migration towards ammonia-enriched areas. Without applying other external field, the directional navigation of the Zn micromotors is realized through the concentration gradient of ammonia.
Firstly, cotton soaked with either NH3·H2O or water was placed on the left side inside a Petri dish, and Zn micromotors were dripped onto the right side. The trajectories of the micromotors were observed and recorded (Fig. 3a). When cotton was soaked with 150 µM NH3·H2O (10 µL), the movement of the Zn micromotors was tracked (Supplementary Movie 2). As shown in Fig. 3b and 3c, the Zn micromotors showed a tendency to move directionally towards the left where the ammonia was located, with an average speed of 1.12 ± 0.73 µm/s and a directionality of 0.24 (Supplementary Table 2) When cotton was soaked with 1500 µM NH3·H2O, the movement process (Supplementary Movie 3) of the Zn micromotors was recorded, and the velocity increased to 1.74 ± 1.33 µm/s, showing directional movement towards the NH3·H2O (Figs. 3d, 3e), with a directivity of 0.21 (Supplementary Table 2). When cotton was soaked in water (Supplementary Movie 4), the statistical analysis of the motion trajectories and direction data were shown in Supplementary Figs. 7, 8. In this case, Zn micromotors lacked directional movement, with an average movement speed of 0.87 ± 0.60 µm/s and directionality of 0.12 (Supplementary Table 2).
The response of Zn micromotors to different chemical attractant (150 µM NH3·H2O, 1500 µM NH3·H2O) (Fig. 3f) was statistically analyzed. Compared with that in water, the speed of Zn micromotors increased by 28.74% in the 150 µM NH3·H2O attractant system and by 100.00% in the 1500 µM NH3·H2O attractant system. Similarly, the chemotaxis directionality (Fig. 3g) of the Zn micromotors increased by 100.00% in the 150 µM NH3·H2O attractant system and 75.00% in the 1500 µM NH3·H2O attractant system, compared with in water. Based on these experimental results, it was determined that the Zn micromotors could demonstrate collective behavior of chemotaxis toward NH3·H2O.
Collective chemotaxis of Zn micromotors in glass channels
To further evaluate the NH3·H2O chemotaxis behavior of Zn micromotors, we designed a Z shaped glass channel for evaluating chemotaxis as shown in Fig. 4a, and the specific parameters were shown in Supplementary Fig. 9. First, phenolphthalein indicator was added to the channel, and a piece of cotton soaked in water (10 µL) was placed in chamber A while a piece of cotton soaked in NH3·H2O (10 µL) was positioned in chamber B. NH3·H2O diffusion was observed (Supplementary Movie 5). NH3·H2O diffused slowly in the channel to form an ammonia concentration gradient. The glass channel was filled with water and the NH3·H2O concentration in chamber B was 150 mM (10 µL). The ammonia diffused slowly to create a concentration gradient. 10 µL of Zn micromotors dispersed in water was placed at the position of C in the middle of the channel (Fig. 4a), and the two positions of the left turn (point D) and the right turn (point E) were selected as observation points. The movement of the Zn micromotors at these two positions was recorded (Supplementary Movies 6 and 7). The moving trajectories of the Zn micromotors at the point D (in the direction of water attractant) were normalized (Supplementary Fig. 10), and the chemotaxis data were statistically analyzed (Supplementary Fig. 11). The results showed that the Zn micromotors lacked directional movement, with an average motion speed of 0.04 ± 0.02 µm/s and directionality 0.02 (Supplementary Table 3). Figure 4b showed the trajectories of the Zn micromotors at point E (in the direction of ammonia attractant). According to statistical analysis, the average velocity of the Zn micromotors increased to 0.14 ± 0.02 µm/s and the directionality increased to 0.06 (Fig. 4c, Supplementary Table 3). The Zn micromotors showed directional motion. Compared with the point D, the average movement speed of the point E increased by 250.00%, and the directionality increased by 200.00% (Fig. 4d). It shows that NH3·H2O can control the group movement direction of Zn micromotors.
To explore the chemotactic principle of Zn micromotors, the products of Zn micromotors reacting with NH3·H2O were first detected by electrospray ionization mass spectrometry (ESI-MS) (Fig. 4e, and Supplementary Figs. 12, 13). After mass spectrometry analysis, the production of target complex ions [Zn(NH3)1](OH)+, [Zn(NH3)2](OH)+ were preliminarily determined. Thus the chemotactic behavior of the Zn micromotors was mainly due to their chemical reaction with NH3·H2O:
Excessive NH3·H2O reacted with Zn in a coordination reaction to form the complex ion. Due to the diffusion of NH3·H2O, a concentration gradient was formed, with higher concentrations closer to chamber B. To validate the mechanism of chemotactic movement of the Zn micromotors towards ammonia, the chemotactic behavior of the Zn micromotors was simulated using finite element analysis software (COMSOL Multiphysics) (Supplementary Methods). The simulation results showed that for the Zn micromotor, the high concentration of ammonia on the right side results in a fast response. The complex ion concentration gradient around the Zn micromotor gradually decreased from 5.39×10− 5 mol/m3 on the right to the left side (Supplementary Fig. 14). It created an electric field in which the right side had a higher potential (99.5 mV) than the left, and the direction of the electric field was from left to right, while the Zn micromotor moved towards a higher potential (Fig. 4f). Therefore, there was a swarming behavior of Zn micromotors moving towards high ammonia concentrations.
Zn micromotors on behavior of TAA induced hyperammonemia mice
Intraperitoneal injection of thioacetamide (TAA) induced persistent hepatocellular injury and fibrosis (Fig. 5a). A model dose of 100 mg/kg was used and the model duration was 6 weeks. Due to pathological conditions such as liver cell damage, ammonia cannot be metabolized smoothly, leading to hyperammonemia. Here Zn micromotors were used for treatment. Due to the fact that the organs responsible for ammonia production and metabolism are the colon and liver, where ammonia accumulates, treatment methods are divided into oral and intravenous injections. Due to liver damage, liver function was impaired, reducing the liver's metabolic function and leading to nutritional imbalances and conditions such as loss of appetite, which resulted in weight loss. After one week of modelling, the weight of control group increased by 0.33% ± 0.79, and the weight of TAA, Zn micromotor (i.v.), and Zn micromotor (i.g.) groups decreased significantly by 3.10% ± 4.03, 2.72% ± 1.01, 1.33% ± 5.09, respectively. After 3 weeks of Zn micromotor treatment, the body weight of TAA group significantly decreased by 6.51% ± 2.82, and those of Zn micromotor (i.v.) and Zn micromotor (i.g.) groups decreased by 0.09% ± 2.82 and 1.60% ± 4.58, respectively. After 7 weeks of Zn micromotor treatment, compared to the previous treatment, control group and Zn micromotor (i.v.) group gained weight by 4.01% ± 7.46 and 4.53% ± 6.22, respectively. The TAA and Zn micromotor (i.g.) groups lost 7.82% ± 4.26 and 5.52% ± 3.23 of body weight, respectively (Fig. 5b).
The liver was stimulated by TAA for a long time, and excessive collagen deposition led to diffuse fibrosis of liver tissue, which destroyed the structure of liver cells, prevented normal metabolism of blood ammonia, and caused an increase in blood ammonia. Abnormally high levels of ammonia in the blood can disrupt neurotransmitter metabolism, adversely affecting the function of the peripheral nervous system. This disruption can decrease the excitability of the neuromuscular junction, causing muscle contractions to lose their coordination. As a result, moving abnormalities such as limb weakness, spasticity, and paralysis may occur. Consequently, we have preliminarily evaluated the moving behavior of mice to understand these effects better.
To assess the exercise capability of mice, a round glass container was prepared to allow free movement (Supplementary Fig. 15). Mice were tracked and filmed for their motion during the week following treatment (Supplementary Movie 8–11). The tracking results are illustrated in Fig. 5c. The total active distance of mice in the control group was 698.19 ± 4.44 cm, and the average speed was 2.33 ± 0.01 cm/s. Compared with the control group, the total distance for TAA group decreased to 57.32 ± 0.35 cm, with an average speed of 0.19 ± 0.001 cm/s. Relative to the TAA group, both the total path and speed of movement in the Zn micromotor treatment groups showed significant improvements. The total distance (583.44 ± 1.54 cm) and average speed (1.94 ± 0.01 cm/s) of the Zn micromotor (i.v.) group increased by 9.18 times and 9.21 times, respectively. The total distance (533.97 ± 1.70 cm) and average speed (1.78 ± 0.01 cm/s) of Zn micromotor (i.g.) group were increased by 8.32 times and 8.37 times, respectively. From the perspective of behavior, the treatment of Zn micromotors can improve the motor dysfunction of hyperammonemic mice.
Effects of Zn micromotors on blood ammonia concentration and liver function in TAA induced hyperammonemia mice
The above experimental results indicated that the mice had developed abnormalities in their behaviour and the model was initially judged to be valid from a behavioural point of view. Subsequently, the degree of liver injury was assessed by measuring the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (ALB), total bilirubin (TBIL) released by the liver into the blood, and the blood ammonia concentration. Additionally, the Zn content in the body was monitored to ensure that its levels were not excessive.
As shown in Fig. 5d, compared with the control group, the blood ammonia concentration in the TAA group significantly increased by 3.05 times, and the Zn micromotor (i.v.) group and Zn micromotor (i.g.) group significantly decreased by 44.59% and 42.13%, respectively, compared with the TAA group. If the liver fails to convert ammonia into urea in time to be excreted from the body, the level of ammonia in the blood will rise, which can be used as an aid to identifying abnormal liver function.
To assess the extent of liver damage caused by high blood ammonia, biochemical tests of liver function were conducted. Compared with the control group, AST and ALT activities were significantly higher in the TAA group increasing by 2.98 and 6.62 times, respectively. The Zn micromotor (i.v.) group and Zn micromotor (i.g.) group demonstrated some improvement after treatment, with significant decreases in AST values by 35.00% and 43.35%, and in ALT values by 64.40% and 68.27%, respectively, compared to the TAA group (Figs. 5e, f). ALB, which is only synthesized in the liver, decreases when the liver is damaged. ALB values in TAA group, Zn micromotor (i.v.) group and Zn micromotor (i.g.) group all decreased to some extent, though ALB values in the Zn micromotor (i.g.) group slightly recovered, increasing by 12.64% compared with the TAA group (Fig. 5g). The TBIL value in TAA group was 0.96 times higher than that in the control group, and the Zn micromotor (i.v.) group and Zn micromotor (i.g.) group were 43.66% and 48.56% lower than that in TAA group, respectively (Fig. 5h). Bile acids, which are synthesized and secreted in the liver, also show abnormal elevations when liver cells are damaged and bile is not excreted properly. Finally, the concentration of Zn in the serum was detected. The Zn concentration in the Zn micromotor (i.v.) group was 0.08 times higher than that in the TAA group, with no significant differences between the other groups (Fig. 5i). According to the above biochemical detection results, the liver damage of TAA group mice was more serious, and the liver damage of Zn micromotor (i.v.) group and Zn micromotor (i.g.) group mice was alleviated.
Zn micromotors alleviate oxidative stress in TAA-induced hyperammoniated mouse liver
Based on the determination of abnormal liver function, we then calculated the ratio of liver to body weight. An abnormal increase in this ratio suggests that the liver may be in a pathological state such as edema, hyperplasia, fibrosis, or fatty liver. Compared with the control group, the liver weight of the other three groups of mice increased abnormally, with the ratio of liver to body weight in the TAA group increasing by 63.03%. The Zn micromotor (i.v.) group and the Zn micromotor (i.g.) group showed a decrease in the ratio of liver to body weight by 21.50% and 21.94%, respectively, compared to the TAA group (Fig. 6a). This indicated an improvement in the abnormal enlargement of the liver.
Oxidative stress, characterized by the excessive accumulation of reactive oxygen species (ROS) in the body, leads to inflammatory infiltration of neutrophils and promotes an inflammatory response in local tissues. Liver tissue was analyzed for oxidative stress markers. Glutathione (GSH), a key scavenger of ROS and the main free radical scavenger in the body, has antioxidative and detoxifying effects. Compared with the control group, the GSH content in the liver of TAA group decreased by 16.05%. Compared with TAA group, GSH content in Zn micromotor (i.v.) group and Zn micromotor (i.g.) group increased by 0.30 times and 0.34 times, and the level was similar to that of normal mice (Fig. 6b). Superoxide dismutase (SOD), a crucial metalloenzyme in the body's antioxidant process, catalyzes the dismutation of superoxide anion radicals into oxygen and hydrogen peroxide. The T-SOD value of TAA group was significantly reduced by 27.98%, while there was no significant difference between Zn micromotor (i.v.) group and Zn micromotor (i.g.) group compared with the control group (Fig. 6c). In human body, MDA is the product of lipid peroxidation by free radicals. It is cytotoxic and can indirectly reflect tissue peroxidation damage. The TAA group showed a significant increase in MDA of 28.5% compared with the control group, and the MDA value of Zn micromotor (i.v.) group was increased, but there was no significant difference with the control group (Fig. 6d). Thus, Zn micromotors can reduce the oxidative stress in liver tissue to a certain extent.
Histopathological observation of liver in TAA induced hyperammonemia mice
Finally, intuitive histological sections were used to examine the liver damage and the degree of fibrosis. Firstly, liver tissue sections were stained with Sirius Red to evaluate the degree of fibroproliferation in liver lesions. The liver tissue of mice in the control group showed no obvious fibroproliferation, and the tissue structure remained intact (Fig. 6e). In contrast, the liver of mice in the TAA group exhibited significant fibroproliferation with pseudolobule formation. Although fibroproliferation was also present in the liver tissue of the Zn micromotor treatment group, the degree of fibroproliferation was reduced, and the liver tissue in the Zn micromotor (i.g.) group exhibited edema. The staining area of Sirius Red on collagen fibers was quantified using ImageJ software, and the area proportion of collagen fibers was calculated (Fig. 6g). The area percentage of collagen fibers in the TAA group was increased by 4.46 times compared to the control group. The area percentage of collagen fibers in the Zn micromotor (i.v.) group and Zn micromotor (i.g.) group were decreased by 38.33% and 31.81%, respectively, compared to the TAA group. This further confirmed that Zn micromotors can mitigate liver injury by reducing the blood ammonia level in vivo.
Secondly, the pathological characteristics of liver tissues were analyzed by H&E staining. In the control group, the liver tissue was structurally intact, the hepatocytes were arranged radially around the central vein, hepatocytes showed mild edema and degeneration, no definite steatosis was found, no obvious fibrous tissue hyperplasia was found in the portal area, and no definite inflammatory cell infiltration was found in the portal area (Fig. 6f). In the TAA group, the cytoplasm appeared eosinophilic with loose red staining, and nuclei were round with occasional binucleation. Severe edema and degeneration of hepatocytes could be seen, ballooning change was seen, no clear steatosis was seen, there was significant fibrous tissue hyperplasia and extensive inflammatory cell infiltration in the portal area, and bridging necrosis was noticeable between the portal areas and the central veins. The structure of the liver plate was unclear, liver sinusoids were narrowed, and slight inflammatory cell infiltration was observed within hepatocytes. In the Zn micromotor (i.g.) group, fibroplasia in the confluent area was reduced compared to the TAA group, and moderate oedema and degeneration of hepatocytes were noted, with reduced ballooning areas. Compared with the TAA group, the Zn micromotor (i.v.) group still showed hepatocyte edema, but no obvious ballooning, and the proliferation of fibrous tissue in the portal area was significantly reduced. Liver fibrosis was evaluated by liver pathology scoring system (Ishak semi quantitative scoring system). The proliferation of fibrous tissue in the Zn micromotors treatment group was notably reduced compared to the TAA group, and the difference was statistically significant (Fig. 6h). The results demonstrated that Zn micromotors could alleviate liver injury caused by high blood ammonia.
Zn micromotors alleviate oxidative stress in cerebral cortex of mice with TAA induced hepatic encephalopathy
Upon the liver function was compromised, the circulating ammonia in the body could not be eliminated timely. Ammonia readily penetrated the blood-brain barrier in molecular form, and its excessive accumulation in the brain led to hepatic encephalopathy43. Ammonia's significant neurotoxicity caused oxidative stress in the brain, resulting in neuronal damage. In the experiments conducted to evaluate hepatic encephalopathy due to hyperammonemia, a portion of cortical brain tissue was collected to detect oxidative stress biomarkers. The GSH level in the brain was reduced by 54.53% in the TAA group compared to the control group. Compared with TAA group, GSH levels in Zn micromotor (i.v.) group and Zn micromotor (i.g.) group increased by 0.76 times and 0.70 times, and the levels were similar to that in control group (Fig. 7a). T-SOD level was significantly decreased by 21.44% in TAA group, while the Zn micromotor (i.v.) group and Zn micromotor (i.g.) group increased by 0.52 times and 0.41 times compared with TAA group, respectively, with no significant difference compared with the control group (Fig. 7b). The MDA value had significantly increased by 33.05% in the TAA group. After treatment, the MDA values in the Zn micromotor (i.v.) and Zn micromotor (i.g.) groups decreased by 30.69% and 27.7%, respectively (Fig. 7c). The above experimental results showed that the abnormal increase of blood ammonia in mice not only accelerated the formation of liver fibrosis, but also further caused a certain degree of damage to the brain. According to the detection results of oxidative stress markers, the treatment with Zn micromotors improved the oxidative stress of cerebral cortex.
Histopathological observations on the brain of mice with TAA induced hepatic encephalopathy
Hepatic encephalopathy caused cerebral edema and increased intracranial pressure, leading to central nervous system dysfunction characterized by cognitive, psychiatric, and motor deficits, accompanied by symptoms of reduced tone in the extremities. Preliminary evidence that hepatic encephalopathy significantly affected the neurological function of the brain was previously demonstrated in a behavioral study (Fig. 5c). TAA group exhibited a significant decrease in moving ability. However, there was recovery of moving function following Zn micromotor treatment.
Then, H&E histopathology was conducted on the cortical and hippocampal regions of the brain (Supplementary Fig. 16). In the control group, the brain tissue structure appeared normal, with neurons and astrocytes in the cerebral cortex and hippocampus being round and their nuclei intact; no obvious pathological edema or vacuolization was observed. The TAA group exhibited inflammatory cell infiltration, and neurons showed marked atrophy, degeneration, and apoptosis, particularly in the cortical areas of the brain. In the hippocampal CA1 and CA3 areas, there was evidence of nucleus regression and mild cell swelling. The Zn micromotor (i.v.) and Zn micromotor (i.g.) groups displayed mild neuronal degeneration in the cortical area of the brain and mild cell swelling in the hippocampal CA3 area. The H&E results indicated that treatment with Zn micromotors significantly improved the nerve injury in the cerebral cortex.
Next, to further investigate the fate of neurons after hepatic encephalopathy caused by hyperammonemia. Nissl staining and H&E staining were utilized to evaluate the damage to neurons. After Nissl staining, neurons that were abnormally atrophic and deeply stained were identified as damaged neurons, often referred to as dark neurons44,45. Neuronal necrosis, a decrease in the number of Nissl bodies, and an increase in dark neurons were observed following neuronal damage. The areas of the cerebral cortex and CA1, CA3 and dentate gyrus (DG) of hippocampus45 were selected to count the existing neurons and dark neurons. From Fig. 7d, it was observed that the neurons in the cortex and hippocampus (CA1, CA3, DG) of the control group were arranged regularly, the Nissl bodies within the neurons were obvious and abundant, and the number of dark neurons was low. In the TAA group, the neurons in the cerebral cortex were noticeably disordered, and the morphology exhibited a certain degree of atrophy. According to statistics, compared with the control group, the number of normal neurons in the cerebral cortex in the TAA group decreased by 47.20%, and the number of dark neurons increased by 111.11% (Fig. 7e). After treatment with Zn micromotors, the arrangement of neurons in the Zn micromotor (i.v.) group were arranged more regularly, and the degree of cell atrophy was restored. The number of normal neurons recovered by 75.94% and the number of dark neurons decreased by 51.43% compared with the TAA group. The number of normal neurons in the Zn micromotor (i.g.) group recovered by 23.58% compared with the TAA group, but the number of dark neurons had no significant difference compared with the TAA group. It was shown that the treatment of Zn micromotors could improve neuronal damage in cerebral cortex, and the effectiveness of intravenous injection is better than that of gastrointestinal administration.
In the hippocampal CA1 area, as shown in the Figs. 7d and 7f, compared with the control group, the Nissl body staining of neurons in TAA group was lighter and the structure was fuzzy. The number of dark neurons significantly increased by 96.84%. Compared with the TAA group, the neurons in the Zn micromotor (i.v.) group were regularly arranged, and the number of dark neurons decreased by 45.45%, and the number of dark neurons in the Zn micromotor (i.g.) group decreased by 47.59%. There was no significant difference in the number of normal neurons in each group in CA1 area. In the hippocampal CA3 area, as shown in the Figs. 7d and 7g, the TAA group showed neuronal edema, light Nissl body staining, and fuzzy structure. According to statistics, the number of dark neurons increased by 36.36%. The Zn micromotor treatment group did not significantly improve the appearance of dark neurons in this area. The hippocampal DG area, as shown in Figs. 7d and 7h, revealed no significant difference in the statistical number of normal neurons among the groups. There was a significant increase of 75.38% in the number of dark neurons in the TAA group compared to the control group. Compared with TAA group, the dark neurons in Zn micromotor (i.v.) group and Zn micromotor (i.g.) group decreased by 33.97% and 32.06%, respectively.
The above results proved that Zn micromotor (i.v.) treatment had a certain protective effect on neurons in cerebral cortex and a certain inhibitory effect on the production of dark neurons in cerebral cortex and hippocampus (CA1, DG). The therapeutic effect of Zn micromotor (i.g.) treatment was slightly less effective than that of Zn micromotor (i.v.), but it also provided a protective effect on neurons in the cerebral cortex and inhibited the production of dark neurons in the hippocampal CA1 area.
Brain edema is also considered an important feature of hepatic encephalopathy. Therefore, the brain weight ratios were calculated (Fig. 6a). Three other groups of mice showed an abnormal increase in brain weight compared to the control group, and the TAA group showed an increase in brain weight ratio of 124.40%. Compared with the TAA group, the brain weight ratio of Zn micromotor (i.v.) group and Zn micromotor (i.g.) group decreased by 31.31% and 32.25%. These results indicated that Zn micromotors improved brain edema after treatment.
Astrocytes are the only cells in the brain that can detoxify ammonia. The abnormally elevated ammonia levels in the brain lead to astrocyte oedema and dysfunction46. Therefore, the cerebral cortex and hippocampus were immunohistochemically labeled with GFAP antibody, and the resulting immunohistochemical maps were measured with ImageJ to calculate the mean optical density value. Results as shown in Supplementary Figs. 17, 18, GFAP expression in the cortical area of the brain was significantly reduced by 43.45% in the TAA group compared to the control group. And significantly increased by 56.20% after Zn micromotor (i.v.) treatment and by 45.57% after Zn micromotor (i.g.) treatment. There was no significant difference in the average optical density between the groups in the hippocampus, indicating that GFAP expression did not change significantly. The results showed that Zn micromotor treatment could control the amount of ammonia entering the brain, thus improving the damage to astrocytes in the cerebral cortex.
Inspired by the autonomous motility and versatility of living organisms in nature, artificial micro/nanomotors are engineered to be applied in biomedical scenario. However, with rapid development in this direction, motion controllability issue is beginning to emerge, or the ability to propel autonomously while ensuring precise targeting. Accurate delivery to the target site avoids accumulation in healthy tissue and unnecessary side effects. Chemotaxis of artificial micro/nanomotors emerges as an effective solution for autonomous collective navigation and in vivo targeted therapy.
To achieve the directional enrichment of artificial micro/nanomotors in vivo, we have developed an artificial micromotor navigation system. The metallic Zn micromotors, are chemically actuated by complexation reaction. With ammonia as chemical attractant, Zn micromotors can chemotactically advance in the direction of high ammonia concentration. For in vitro model, the Zn micromotor swarm followed the ammonia concentration gradient, achieving long-distance autonomous migration toward an ammonia source.
For in vivo applicability, we chose the TAA-induced liver fibrosis model mice. Upon hepatocytes injury in TAA, the ability to scavenge ammonia is greatly reduced (3.05 times increased in blood ammonia concentration compared to control group), leading to ammonia accumulation in the liver, which aggravated liver fibrosis and induces hyperammonemia. Excess ammonia penetrates the blood-brain barrier in a non-ionic form and accumulates in the brain, causing oxidative stress, neuronal necrosis and hepatic encephalopathy.
Clinical prognosis of liver fibrosis and the treatment of hepatic encephalopathy focus on reducing blood ammonia. With Zn micromotors exhibit chemotaxis towards ammonia, we have designed them to target the colon (ammonia production site) and liver (ammonia metabolism site) through oral or intravenous injection, respectively. The micromotors locate and target areas of high ammonia concentration, and trap ammonia during their movement via coordination reactions, forming the complex ions [Zn(NH3)1](OH)+ and [Zn(NH3)2](OH)+, which could be excreted via kidney. This process further reduces the concentration of ammonia in the circulation system (42.13% − 44.59% decrease). Moreover, Zn itself can assist the liver in ammonia metabolism47–49. The system prevents further aggravation of liver fibrosis and reduce the deposition of collagen fibers by lowering ammonia accumulation in the liver (38.33% reduction in fibrotic area for intravenous treatment group, 31.81% reduction for oral treatment group). With minimized ammonia penetration into the brain, the number of normal neurons in the cerebral cortex experienced an increase (75.94% increase after intravenous injection and 23.58% increase after oral treatment). Behaviorally, the treatment of Zn micromotors also improved the moving dysfunction of mice. After intravenous injection of Zn micromotors, the average speed increased by 9.21 times compared with the TAA group.
In this study, we propose for the first time a generalizable chemotaxis strategy through coordination reaction. Through the interplay (complexation) between imposed external ammonia gradient and the self-generated Zn ion gradient of the active material, long-distance and collective directional navigation is realized. Upon reaching target position, the hybrid interface trap excess ammonia to generate complex ions, thus reshaping local pathological microenvironment and reducing the toxicity of ammonia. This adaptive system represents an intelligent chemotactic navigation therapy, providing a promising approach for green and precision therapy in the future.
Polycarbonate film (PC film) was purchased from Whatman (Shanghai, China). Boric acid (H3BO3) and aqueous ammonia (NH3·H2O) were purchased from Macklin (Shanghai, China). Zn sulfate heptahydrate (ZnSO4·7H2O) was purchased from Aladdin (Shanghai, China). Ethanol and dichloromethane (CH2Cl2) were purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China). Thioacetamide (TAA) was purchased from MeilunBio (Dalian, China). GFAP antibody was purchased from Proteintech Group (Wuhan, China). Blood Ammonia Content Assay Kit was purchased from Beijing Solarbio Science&Technology Co.,Ltd. (Beijing, China). Alanine Aminotransferase (ALT) assay kit, Aspartate Aminotransferase (AST) assay kit, and Albumin (ALB) assay kit were purchased from Rayto (Shenzhen, China). Total bilirubin assay kit was purchased from Changchun Huili biotech co., ltd (Changchun, China). Zn Assay Kit and Glutathione Peroxidase (GSH-PX) assay kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Total Superoxide Dismutase (T-SOD) Assay Kit and Malondialdehyde (MDA) assay kit were purchased from Servicebio (Wuhan, China).
Fabrication of Zn micromotors
Zn micromotors were fabricated by electrochemical deposition using membrane plate method. The three-electrode system was selected, and the corresponding three electrodes were working electrode (copper electrodeposition support, 13 mm PEEK), reference electrode (Ag/AgCl), and counter electrode (platinum wire). Polycarbonate membrane used for formwork. First, a 100 nm thick gold layer was evaporated on one side of the polycarbonate film using an electron beam evaporation wave test DE400 for conductivity and to close the single-sided film aperture. The polycarbonate film (gold layer) was covered on the working electrode for electrochemical deposition using an electrochemical workstation. At room temperature, a mixed solution of 0.2 M ZnSO4·7H2O and 0.1 M boric acid was used for electrodeposition at a constant voltage of − 1.23 V for 15 min. After the preparation, the gold layer on the membrane surface was removed by physical friction, and then the polycarbonate membrane template was dissolved with dichloromethane. The Zn micromotors were dispersed in water for storage.
Characterization of Zn micromotors
Zn micromotors were dispersed with ethanol on silicon wafers, dried and characterised by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) (Hitachi, regulus 8230). The movement of Zn micromotors in water were recorded using Nikon inverted microscope (Nikon, Ti2-A) and NIS-Element Viewer software.
Animal Experiments
Associated animal use protocols have been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Sun Yat sen University. Male Balb/c mice (6–8 weeks) were provided by the Laboratory Animal Centre of Sun Yat-sen University and were housed according to standard procedures at temperature (20–25℃), relative humidity (40–60%) and in a specific pathogen-free environment. Food and water were freely available for five days prior to the start of the experiment to allow for environmental acclimatisation.
Experimental design
After five days of adaptation, refer to the experimental protocol. Twenty mice were randomly divided into the following four groups: (1) The control group was given 0.9% (w/v) normal saline (5 ml/kg, i.p., 3 times per week for 6 weeks); (2) Thioacetamide (TAA) model group (100 mg/kg, i.p., 3 times per week for 6 weeks); (3) Zn micromotors (i.v.) group (TAA 100 mg/kg, i.p., 3 times per week for 6 weeks; Zn micromotors, 10 µL/g, i.v., was given from the second week, 3 times per week for 8 weeks); (4) Zn micromotors (i.g.) group (TAA 100 mg/kg, i.p., 3 times per week for 6 weeks; Zn micromotors, 10 µL/g, i.g., was given from the second week, 2 times per week for 8 weeks) was given from the second week. Body weight was recorded before each administration. Mouse movement tracking was performed after each administration in the last week. Two hours after the last administration, the mice were euthanized and blood samples were collected. Liver and brain were harvested, weighed and a portion used to prepare tissue homogenate and the remainder preserved for histopathological examination.
Body weight change
The formula for calculating the percentage change in mouse weight is: (Body weight of the next day - Body weight of the previous day) × 100/ Body weight of the previous day.
Measurement of liver brain body weight index
The liver and brain were weighed and recorded immediately after removal.
The formula of liver body weight index (LBWI) and brain body weight index (BBWI) are: LBWI= (liver weight / body weight) and BBWI= (brain weight / body weight), respectively.
Behavioral observation
In a quiet environment, the mice were put into a round glass container at the bottom of the open field box, and the movie was taken and timed for 5 min at the same time. The voluntary movements of the mice were observed and recorded, the trajectories were plotted using the Tracker software, and the speeds of movement were calculated.
Biochemical assay
Blood was collected from the orbits and allowed to stand at room temperature (25°C) for 1 h, then centrifuged at 2800 rpm for 15 min at 4°C. Transfer the supernatant, i.e. serum, to the corresponding labeled sample tube for storage, and store the serum at − 80 ℃ after collection, waiting for subsequent detection. The contents of blood ammonia, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (ALB), total bilirubin, Zn in serum were determined according to the instructions of the biochemical detection kit. Take appropriate amount of liver and cerebral cortex tissue to prepare tissue homogenate, and determine the contents of glutathione peroxidase (GSH-Px), total superoxide dismutase (T-SOD) and malondialdehyde (MDA) in tissue homogenate according to the instructions of biochemical detection kit.
Histopathological examination
The liver and brain of mice were taken, washed with PBS (pH 7.4), and the tissues were fixed in 4% paraformaldehyde (PFA). Then they were dehydrated and embedded in paraffin, sliced at approximately 5 µm thickness using a pathology slicer (Leica Biosystems, Germany), and the slices were fixed onto microscope slides.
H&E staining analysis: Each group of liver and brain tissue sections were stained with haematoxylin and eosin (H&E). After staining, tissue images of liver and brain were taken under a microscope. The pathological changes in the tissues were observed.
Sirius red staining analysis: Liver tissues of each group were stained with Sirius red staining solution, and after staining, images of liver tissues were taken using a microscope, and the images were processed using ImageJ software to statistically measure the area of Sirius red collagen fibres, which was used to observe the degree of liver fibrosis in mice.
Nissl staining analysis: Brain tissues of each group were stained with Nissl staining solution. After staining, the brain tissue images were taken using a microscope, and the number of normal neurons and dark neurons in the cerebral cortex and hippocampus (CA1, CA3, DG) were counted using imageJ software. It was used to observe the neuronal damage in the cerebral cortex and hippocampus of mice.
Immunohistochemistry analysis
The basic procedure is to repair the antigen with repair solution. Sections were incubated in 3% H2O2 solution for 20 min at room temperature to block endogenous peroxidase. After blocking with 3% bovine serum albumin BSA for 30 min, rabbit anti-mouse GFAP primary antibody was added and incubated overnight at 4°C. The next day, the tissue was labelled with horseradish peroxidase (HRP) secondary antibody and incubated for 1 hour at room temperature. Brain tissue images were taken using a microscope after DAB (3,3'-diaminobenzidine) colour development, and semi-quantitative statistics were performed on the sections using ImageJ software using the average optical density values expressed for the observation The extent of damage to astrocytes in the cerebral cortex and hippocampus (CA1, CA3, DG) was observed.
Statistical analysis
Data in this paper were statistically analysed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) and Origin 8.5 (OriginLab, Northampton, Massachusetts, USA). Values are expressed as mean ± standard deviation (SD). Each independent experiment had at least 5 sample replicates and comparisons between experimental groups were statistically analysed using the t-test. p < 0.05 was considered a statistically significant difference.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No. 21805318, 51973241, and 22175083), Guangdong Provincial Science Foundation for Distinguished Young Scholars (Grant No. 2018B030306007) and National Key Research and Development Program (2022YFA1206900).
Author contributions
Y.T. and F.P. conceptualized the study and acquired funds. Y.F., C.G., B.C. and M.D. conducted experiments. Y.F. and D.D. carried out data analyses. Q.L. and J.R. provided experimental expertise. Y.F. and C.G. wrote and edited the manuscript. D.A.W. reviewed the manuscript.
Conflict of Interest
The authors declare no conflict of interest.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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There is NO Competing Interest.
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