Light-powered phagocytic macrophage microrobot (phagobot): both in vitro and in vivo

Abstract

Micro/nanorobots based on immune cells show great potential for addressing challenging biological and biomedical conditions. However, their powerful innate immune functions, particularly the phagocytosis capabilities, remain a big challenge to fully leverage with the current designs of immune cell-based microrobots. Herein, we report a light-powered phagocytic macrophage microrobot (phagobot), which is capable of robotic navigation toward specific foreign bio-threats and executing precise phagocytosis of these targeted entities under light control. Without genetic modification or nanoengineering of macrophages, the phagobot's "wake-up" program is achieved through direct activation of a resting-state macrophage by a tightly focused near-infrared (NIR) light beam. The phagobot exhibits robotic steering and directional navigation controlled by optical manipulation of the extended pseudopodia within the activated macrophage. It can further execute targeted phagocytic clearance tasks via engulfing various foreign bio-threats, including nanoplastics, microbials, and cancer cell debris. Notably, the phagobot can be constructed in a living larval zebrafish through optical activation and manipulation of the endogenous macrophage, which also exhibits controllable navigation and targeted phagocytic capabilities in vivo. With the intrinsic immune functions of macrophages, our light-powered phagobot represents a novel form of intelligent immune cell-based microrobots, holding many new possibilities for precise immune regulation and treatment for immune-related diseases.

Fig. 1. Overview of light-controlled macrophage phagobot.

a Schematic illustration of light-controlled phagobot for targeted phagocytic clearance of bio-threats. The wake-up program for phagobot is realized by localized optothermal stimulation of a resting macrophage through NIR light micro-irradiation. The steering and navigation of the phagobot can be flexibly controlled via optical manipulation of the extended filopodia in the activated macrophage, and the phagobot can be further controlled to execute immune clearance tasks via phagocytosis of various bio-threats with different sizes. b Time-lapse images showing the activation of a resting macrophage through NIR light micro-irradiation. Red dots indicate the position of the NIR light beam, and yellow arrows indicate the extended filopodia in the activated macrophage. c Changes in nthe umber of filopodia in (b) over time during NIR light micro-irradiation

Fig. 2. Activation of phagobot through NIR light micro-irradiation.

a Mechanism of photothermal activation of phagobot by NIR light micro-irradiation. b Simulated temperature distribution in a macrophage exposed to NIR light micro-irradiation at an optical power of 60 mW. c Calculated temperature of irradiated site in cells at different irradiation power. d Intracellular Ca²⁺ levels in macrophages stained with Fluo-4 AM fluorescent probe (I) before and (II) after exposure to NIR light micro-irradiation for 3 min. III and IV, suppression of TRPM2 activation by econazole as negative control. e Quantitative statistics of intracellular Ca²⁺ level over time at different irradiation power. f Mitochondrial membrane potential (MtMP) analysis of macrophage following NIR light micro-irradiation using fluorescent probe TMRM. The cell was stimulated by micro-irradiation for 3 min, then continuously observed for another 12 min. g Quantitative statistics of changes in MtMP over time. h Intracellular ATP levels in control and stimulated macrophages with the live-cell ATP dye pCMV-AT1.03. i Quantitative statistics of intracellular ATP levels over time during micro-irradiation at different laser power. j Intracellular ROS level in a macrophage stained with CM-H₂DCFDA fluorescent probe. k Quantitative statistics of changes in intracellular ROS levels over time at different irradiation power. n = 5–6/group. Scale bar for all fluorescent images: 10 μm

Fig. 3. In vitro motion control of phagobot.

a Phagobot steering. I: schematic illustration of controllable rotation of phagobot steered by programmed annular scanning focused light beam. II: time-lapse image sequences of the steering/rotation of the phagobot. Red dots indicate the position of light beam, red dashed curves indicate the predesigned light beam trajectory, blue lines indicate the steering angle, and yellow dots point to the extended filopodia. b Angular velocity (ω) of phagobot steering/rotation regulated by varying optical power. n = 5–6/group. c Phagobot navigation. I: schematic illustration of directed navigation of phagobot controlled by programmable optical force. II: time-lapse image sequences of directed navigation of the phagobot toward a targeted cell. Green dashed circles and blue dashed lines indicate the initial position and the moving trajectory of the phagobot, respectively. d Quantitative statistics of linear motion velocity of phagobot as a function of laser power. n = 5–6/group. e Composite motion task of directional steering and navigation. I: schematic illustration of directional steering and navigation of phagobot controlled by programmed composite light beam. II: time-lapse image sequences of the round-trip movement with U-turn action of the phagobot. Scale bar: 10 μm

Fig. 4. Execution of targeted phagocytic tasks.

a Schematic showing targeted phagocytosis of foreign bio-threats by light-controlled phagobot. b Time-lapse image sequences showing the phagocytosis process of a yeast cell by phagobot. Red dots indicate the position of light beam, red dashed curves indicate the predesigned light beam trajectory, white and yellow arrows indicate the targeted yeast cell and extended filopodia in the phagobot, respectively. Scale bar: 10 μm. c Fluorescent images showing the phagocytosis of a 500-nm polystyrene (PS) nanoparticle by phagobot. Red: PS nanoparticle, blue: cell nucleus of macrophage, green: actin cytoskeleton. Scale bar: 5 μm. d Sequential phagocytosis of multiple S. aureus bacteria by phagobot in a programmable manner. e Average rate of engulfment (min ± SD) of a single bio-threat of different sizes by the phagobot. n = 5–6/group. Scale bar: 10 μm. f Number of phagocytosed bacteria by phagotbot over time by varying laser powers. n = 5–6/group

Fig. 5. Construction and navigation control of phagobot for targeted phagocytosis in a living larval zebrafish.

a Optical microscopy (I) and fluorescence images (II) of larval zebrafish with fluorescence-labeled macrophages. Scale bar: 500 μm. b Optical microscopic images for observing single fluorescence-labeled macrophages in the intestinal cavity tissues of larval zebrafish. Scale bar: 10 μm. c Navigation of a phagobot along a complex trajectory in the intestinal cavity tissue. Red dots and red arrows indicate the position and moving direction of light beam, respectively. Green dashed circles and blue dashed lines indicate the initial position and the moving trajectory of the phagobot, respectively. d Migration velocity of phagobot over time in (c). In vivo e navigation velocity and f locomotion distance of the phagobot as a function of laser power. n = 5–6/group. g Optical microscopic images for navigating the phagobot to eliminate targeted cell debris. Yellow dashed circles indicate the targeted cell debris. h Measured heart rates of zebrafish stimulated by various laser powers. n = 5 hearts