Recently, tremendous progress in synthetic micro/nanomotors in diverse environment has been made for potential biomedical applications. However, existing micro/nanomotor platforms are inefficient for deep tissue imaging and motion control in vivo. Here, we present a photoacoustic computed tomography (PACT)–guided investigation of micromotors in intestines in vivo. The micromotors enveloped in microcapsules are stable in the stomach and exhibit efficient propulsion in various biofluids once released. The migration of micromotor capsules toward the targeted regions in intestines has been visualized by PACT in real time in vivo. Near-infrared light irradiation induces disintegration of the capsules to release the cargo-loaded micromotors. The intensive propulsion of the micromotors effectively prolongs the retention in intestines. The integration of the newly developed microrobotic system and PACT enables deep imaging and precise control of the micromotors in vivo and promises practical biomedical applications, such as drug delivery.

( A ) Schematic of the PAMR in the GI tract. The MCs are administered into the mouse. NIR illumination facilitates the real-time PA imaging of the MCs and subsequently triggers the propulsion of the micromotors in targeted areas of the GI tract. ( B ) Schematic of PACT of the MCs in the GI tract in vivo. The mouse was kept in the water tank surrounded by an elevationally focused ultrasound transducer array. NIR side illumination onto the mouse generated PA signals, which were subsequently received by the transducer array. (Inset) Enlarged view of the yellow dashed box region, illustrating the confocal design of light delivery and PA detection. US, ultrasound; CL, conical lens; DAQ, data acquisition system. ( C ) Enteric coating prevents the decomposition of MCs in the stomach. ( D ) External CW NIR irradiation induced the phase transition and subsequent collapse of the MCs on demand in the targeted areas and activated the movement of the micromotors upon unwrapping from the capsule. ( E ) Active propulsion of the micromotors promoted retention and cargo delivery efficiency in intestines.

Here, we describe a PACT-guided microrobotic system (PAMR), which has accomplished controlled propulsion and prolonged cargo retention in vivo ( Fig. 1A and movie S1). Because of high spatiotemporal resolution, noninvasiveness, molecular contrast, and deep penetration, PACT provides an attractive tool to locate and navigate the micromotors in vivo ( Fig. 1B ) ( 18 – 20 ). Ingestible Mg-based micromotors were encapsulated in enteric protective capsules to prevent reactions in gastric acid and allow direct visualization by PACT ( Fig. 1, A to C ). PACT monitored the migration of micromotor capsules (MCs) in intestines in real time; continuous-wave (CW) near-infrared (NIR) light irradiation induced phase transition of microcapsules and triggered propulsion of the micromotors ( Fig. 1D ); autonomous and efficient propulsion of the micromotors enhanced the retention in targeted areas of the GI tract ( Fig. 1E ). We believe that the proposed integrated microrobotic system will substantially advance GI therapies.

Drug delivery through the gastrointestinal (GI) tract serves as a convenient and versatile therapeutic tool, owing to its cost effectiveness, high patient compliance, lenient constraint for sterility, and ease of production ( 22 , 23 ). Although oral administration of various micro/nanoparticle-based drug delivery systems has been demonstrated both to survive the acidic gastric environment and to diffuse into the intestines, drug absorption is still inefficient because of the limited intestinal retention time ( 24 ). A large number of passive diffusion–based targeting strategies have been explored to improve the delivery efficiency, but they suffer from low precision, size constraint, and specific surface chemistry ( 25 ). With precise control, microrobotic drug delivery systems can potentially achieve targeted delivery with long retention times and sustainable release profiles, which are in pressing need ( 26 ). Because of the lack of imaging-guided control, there is no report yet for precisely targeted delivery using micromotors in vivo ( 14 ). In addition, biodegradability and biocompatibility are required, and an ideal microrobotic system is expected to be cleared safely by the body after completion of the tasks ( 5 , 27 , 28 ).

To date, optical imaging is widely used for biomedical applications owing to its high spatiotemporal resolution and molecular contrasts. However, applying conventional optical imaging to deep tissues is hampered by strong optical scattering, which inhibits high-resolution imaging beyond the optical diffusion limit (~1 to 2 mm in depth) ( 16 ). Fortunately, photoacoustic (PA) tomography (PAT), detecting photon-induced ultrasound, achieves high-resolution imaging at depths that far exceed the optical diffusion limit ( 17 ). In PAT, the energy of photons absorbed by chromophores inside the tissue is converted to acoustic waves, which are subsequently detected to yield high-resolution tomographic images with optical contrasts. Leveraging the negligible acoustic scattering in soft tissue, PAT has achieved superb spatial resolution at depths, with a depth-to-resolution ratio of ~200 ( 18 ), at high imaging rates. As a major incarnation of PAT, PA computed tomography (PACT) has attained high spatiotemporal resolution (125-μm in-plane resolution and 50-μs frame −1 data acquisition), deep penetration (48-mm tissue penetration in vivo), and anatomical and molecular contrasts (text S1) ( 19 – 21 ). With all these benefits, PACT shows promise for real-time navigation of micromotors in vivo for broad applications, particularly drug delivery.

Micro- and nanorobots that can be navigated into hard-to-reach tissues have drawn extensive attention for the promise to empower various biomedical applications, such as disease diagnosis, targeted drug delivery, and minimally invasive surgery ( 1 – 6 ). Chemically powered motors, in particular, show great potential toward in vivo application owing to their autonomous propulsion and versatile functions in bodily fluids ( 7 – 11 ). However, imaging and control of micromotors in vivo remain major challenges for practical medical investigations, despite the substantially advanced development of micromotors ( 12 – 15 ). The ability to directly visualize the dynamics of micromotors with high spatiotemporal resolution in vivo at the whole-body scale is in urgent demand to provide real-time visualization and guidance of micromotors ( 14 ). In addition to high spatiotemporal resolution, the ideal noninvasive micromotor imaging technique should offer deep penetration and molecular contrasts.

RESULTS

Fabrication of the MCs The fabrication of MCs mainly consists of two steps: the fabrication of Mg-based micromotors (see fig. S1 and Materials and Methods) and the formation of MCs (see fig. S2 and Materials and Methods). In the first step, Mg microparticles with a diameter of ~20 μm were dispersed onto glass slides, followed by the deposition of a gold layer, which facilitates the autonomous chemical propulsion in GI fluids and enhances PA contrast of the micromotors. An alginate hydrogel layer was coated onto the micromotors by dropping aqueous solution containing alginate and drugs (e.g., doxorubicin) on the slides. A parylene layer, acting as a shell scaffold that ensures the stability during propulsion, was then deposited onto the micromotors. Figure 2A illustrates a fabricated spherical micromotor (~20 μm in diameter). A small opening (~2 μm in diameter), attributed to the surface contact of the Mg microparticles with the glass slides during various layer coating steps, acts as a catalytic interface for gas propulsion in the intestinal environment. Next, the micromotors were encapsulated into the enteric gelatin capsules by the emulsion method (fig. S2). Green fluorescence from the fluorescein isothiocyanate–labeled bovine serum albumin (FITC-albumin) and red fluorescence from doxorubicin (DOX) were observed from the micromotors (see fig. S3 and Materials and Methods) and the MCs (see fig. S4 and Materials and Methods), confirming a successful drug loading. The size of MCs could be varied by changing the speed of magnetic stirring (fig. S5). The microscopic images in Fig. 2B show three MCs with diameters of 68, 136, and 750 μm. Fig. 2 Characterization of the MCs. (A) SEM image of an ingestible micromotor. Scale bar, 10 μm. (B) Microscopic images of the MCs with different sizes. Scale bars, 50 μm. (C) PACT images of Mg particles, blood, and MCs in silicone rubber tubes with laser wavelengths at 720, 750, and 870 nm, respectively. Scale bar, 500 μm. (D) PACT spectra of MCs (red line), blood (blue line), and Mg particles (black line). (E and F) PACT images (E), the corresponding PA amplitude (F) of the MCs with different micromotor loading amounts, and the dependence of the PA amplitude on the fluence of NIR light illumination [inset in (F)]. Scale bar, 500 μm (E). (G) Dependence of PA amplitude of the MCs (red line) and blood (black line) on the depth of tissue and the normalized PA amplitude and fluorescence intensity of the MCs under tissues (inset). Norm., normalized; amp., amplitude; Fl., fluorescence; int., intensity. Error bars represent the SDs from five independent measurements. For deep tissue imaging in vivo, it is crucial that the MCs have a higher optical absorption than the blood background. To evaluate the PA imaging performance of the MCs, we measured the PA amplitudes of the MCs, whole blood, and bare Mg particles (see Materials and Methods). NIR light experiences the least attenuation in mammalian tissues, permitting the deepest optical penetration. As shown in Fig. 2C, the MCs exhibit strong PA contrast in the NIR wavelength region, ranging from 720 to 890 nm. To assess quantitatively the optical absorption of the MCs, we extracted amplitude values from the above PA images and subsequently calibrated them with optical absorption of hemoglobin (29, 30). At the wavelength of 750 nm, the MCs display the highest PA amplitude of 15.3 (Fig. 2D). The bare Mg particles display a similar PA spectrum, with a lower PA peak with an amplitude of 10.0 at 750 nm. The difference due to the Au layer is expected to significantly improve the imaging sensitivity in the NIR wavelength region (Fig. 2D) (31, 32). In addition, the approximate threefold increase in PA amplitudes of the MCs compared with that of the whole blood provides sufficient contrast for PACT to detect the MCs in vivo using 750-nm illumination. To evaluate the stability of the MCs under pulsed NIR PA excitation, we measured the PA signal fluctuation of the MCs during PA imaging (fig. S6). The negligible changes in the PA signal amplitude during the operation suggest a remarkably high photostability of the MCs. Figure 2 (E and F) shows the PA images and the corresponding PA amplitudes of single MCs with different concentrations of micromotors. The dependence of the PA amplitude on the NIR light fluence (i.e., energy per area) was also investigated (see Materials and Methods). As expected, the PA amplitude of the micromotors almost linearly increases with the NIR light fluence (Fig. 2F, inset). We also studied the maximum detectable depth of MCs using PACT (see Fig. 2G and Materials and Methods). The micromotors showed markedly decreased fluorescence intensity when covered by thin tissues (0.7 to 2.4 mm in thickness) and became undetectable quickly [Fig. 2G (inset) and fig. S7]. By contrast, PACT could image the micromotors inside tissue as deep as ~7 cm (Fig. 2G), which reveals that the key advantage of PACT lies in high spatial resolution and high molecular contrast for deep imaging in tissues (19).

Characterization of the dynamics of the PAMR in vitro The high optical absorption of the MCs empowers the PAMR as a promising in vivo imaging contrast agent. To evaluate the dynamics of the PAMR, we conducted the PA imaging experiments initially in vitro, where silicone rubber tubes modeled intestines (see Materials and Methods). The tubular model intestine was sandwiched in chicken breast tissues (Fig. 3A). The PA time-lapse images in Fig. 3B and movie S2 illustrate real-time tracking of the migration of an injected MC in the model intestine. Fig. 3 Characterization of the dynamics of the PAMR. (A and B) Schematic (A) and time-lapse PACT images in deep tissues (B) illustrating the migration of an MC in the model intestine. Scale bar, 500 μm. The thickness of the tissue above the MC is 10 mm. (C to E) Schematic (C) and time-lapse microscopic images (D and E) showing the stability of the MCs in gastric acid and intestinal fluid (D) without CW NIR irradiation and the use of CW NIR irradiation to trigger the collapse of an MC and the activation of the micromotors (E). Scale bars, 50 μm (D and E). In addition to tracking and locating the MCs, propulsion of the micromotors upon unwrapping from the microcapsules could be activated on demand with high-power CW NIR irradiation (see Fig. 3C and Materials and Methods). Because of the enteric coating and gelatin encapsulation, the MCs showed long-term stability in both gastric acid and intestinal fluid (Fig. 3D and fig. S8). The Au layer of the micromotors could effectively convert NIR light to heat, resulting in a gel-sol phase transition of the gelatin-based capsule followed by the release of the micromotors. Such CW NIR–triggered disintegration of the MCs usually occurred within 0.1 s. Therefore, CW NIR irradiation could activate autonomous propulsion of the micromotors (Fig. 3E and movie S3). Such a photothermal effect also significantly accelerated the Mg-water chemical reactions and thus enhanced the chemical propulsion of the micromotors. As shown in fig. S9 and movie S4, the micromotors exhibited efficient bubble propulsion in various biofluids. Further quantitative analysis indicates that the velocities of the micromotors were 45 and 43 μm s−1 in phosphate-buffered saline (PBS) solution and the model intestinal fluid, respectively. Note that bare Mg particles have negligible propulsion in neutral media (i.e., intestinal fluid) and disordered propulsion in acidic condition (see fig. S10 and Materials and Methods). The highly efficient propulsion in the targeted areas in intestines provides a mechanical driving force to enhance retention and delivery. The required NIR power can be potentially adjusted by controlling the synthesis process and composition of the MCs. Other triggering mechanisms in biomedicine, such as magnetic or ultrasonic fields, can also be used to activate propulsion of the micromotors (33).

Dynamic imaging of the PAMR in vivo The movement of a swarm of MCs was monitored in vivo by PACT (see Materials and Methods). The MCs were dispersed in pure water and then orally administered into 5- to 6-week-old nude mice. The mice were subsequently anesthetized, and the lower abdominal cavity was aligned with the imaging plane of the ultrasonic transducer array for longitudinal imaging (Fig. 1B). PACT images were captured at a frame rate of 2 Hz for ~8 hours (in Fig. 4A and movie S5, the blood vessels and background tissues are shown in gray, and MCs in intestines are highlighted in color). During the imaging period of the first 6 hours, the MCs migrated ~1.2 cm, roughly 15% of the length of the entire small intestine. After 5 hours, the PA signals of some MCs faded away as they moved downstream in intestines that were outside of the imaging plane. The moving speed of the swarm MCs in the intestines and the movements induced by respiratory motion were quantified (Fig. 4, B to D, and fig. S11). As shown in Fig. 4 (B to D), the abrupt motion caused by respiration was much faster than actual migration of the MCs. Despite the respiration-induced movement, PACT could distinguish the signals from the slowly migrating MCs in the intestines (see Materials and Methods). These results indicate that PACT could precisely monitor and track the locations of the MCs in deep tissues in vivo. Fig. 4 PACT evaluation of the PAMR dynamics in vivo. (A) Time-lapse PACT images of the MCs in intestines for 7.5 hours. The MCs migrating in the intestine are shown in color; the mouse tissues are shown in gray. Scale bar, 2 mm. (B and C) Movement displacement caused by the migration of the MCs in the intestine (B) and by the respiration motion of the mouse (C). (D) Comparison of the speeds of the MC migration and the respiration-induced movement. Error bars represent the SDs from three independent measurements.