DOI: 10.5061/dryad.2rbnzs7x9
title: Biohybrid microrobots regulate colonic cytokine and epithelium barrier in inflammatory bowel disease

description: Cytokines have been identified as key contributors to the development of
 inflammatory bowel disease (IBD), yet conventional treatments often prove
 inadequate and carry substantial side effects. Here, we show an innovative
 biohybrid robotic system, termed ‘algae-MΦNP-robot’, for addressing IBD by
 actively neutralizing colonic cytokine levels. Our approach combines
 moving green microalgae with macrophage membrane-coated nanoparticles
 (MΦNPs) to efficiently capture pro-inflammatory cytokines ‘on-the-fly’.
 The dynamic algae-MΦNP-robots outperform static counterparts by enhancing
 cytokine removal through continuous movement, better distribution, and
 extended retention in the colon. This system is encapsulated within an
 oral capsule, which shields it from gastric acidity and ensures
 functionality upon reaching the targeted disease site. The resulting
 algae-MΦNP-robot capsule effectively regulates cytokine levels,
 facilitating the healing of damaged epithelial barriers. It shows markedly
 improved prevention and treatment efficacy in a mouse model of IBD and
 demonstrates an excellent biosafety profile. Overall, our biohybrid
 algae-MΦNP-robot system offers a promising and efficient solution for IBD,
 addressing cytokine-related inflammation effectively.
description: The dataset was the collection of the data which were used for
 Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, and Figure 6. The
 detailed information of each data collection was included in the
 'Materials and Methods' section of the main
 manuscript.
description: # Biohybrid microrobots regulate colonic cytokine and epithelium barrier
 in inflammatory bowel disease
 [https://doi.org/10.5061/dryad.2rbnzs7x9](https://doi.org/10.5061/dryad.2rbnzs7x9) We have submitted our raw data about Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, and Figure 6 (**adl2007_Science Robotics**). ## Description of the data and file structure **Descriptions:** **Main abbreviations:** **MΦNPs**: macrophage membrane-coated PLGA nanoparticles. **Algae-MΦNP-robot**: macrophage membrane-coated PLGA nanoparticles loaded green algae microrobots. **Static algae-MΦNPs**: macrophage membrane-coated PLGA nanoparticles loaded green algae without flagella. **Bare algae**: bare green algae. **Algae-MΦNP-robot capsule**: macrophage membrane-coated PLGA nanoparticles loaded green algae microrobots encapsulated into the modified Size M mice capsule. **Static algae-MΦNPs capsule**: macrophage membrane-coated PLGA nanoparticles loaded green algae without flagella encapsulated into the modified Size M mice capsule. **Water / PBS**: mice pre-treatment with water then oral administration with 1X PBS. **3% DSS / Algae-MΦNP-robot Capsule**: mice pre-treatment with 3% w/v dextran sodium sulfate then oral administration with macrophage membrane-coated PLGA nanoparticles loaded green algae microrobots encapsulated into the modified Size M mice capsule. **3% DSS / PBS**: mice pre-treatment with 3% w/v dextran sodium sulfate then oral administration with 1X PBS. **3% DSS / Static algae-MΦNPs Capsule**: mice pre-treatment with 3% w/v dextran sodium sulfate then oral administration with macrophage membrane-coated PLGA nanoparticles loaded green algae without flagella encapsulated into the modified Size M mice capsule. **3% DSS / Algae-MΦNP-robot**: mice pre-treatment with 3% w/v dextran sodium sulfate then oral administration with macrophage membrane-coated PLGA nanoparticles loaded green algae microrobots. **Water / Algae-MΦNP-robot Capsule**: mice pre-treatment with water then oral administration with macrophage membrane-coated PLGA nanoparticles loaded green algae microrobots encapsulated into the modified Size M mice capsule. **ELISA**: enzyme-linked immunosorbent assay. ### Each Figure description: ·       **Figure 1D**: Speed of algae-MΦNP-robot in artificial colonic fluid (ACF) at room temperature (22 °C) or body temperature (37 °C); y-axis: motion speed of algae-MΦNP-robot in µm/s, x-axis: the time after transferring algae-MΦNP-robot in artificial colonic fluid (ACF) in hours at room temperature (22 °C) or body temperature (37 °C); each point includes 100 independent datapoints (n = 100). ·       **Figure 1E**: Motile ratio of algae-MΦNP-robot in artificial colonic fluid (ACF) at room temperature (22 °C) or body temperature (37 °C); y-axis: the motile percentage of algae-MΦNP-robot, x-axis: the time after transferring algae-MΦNP-robot in artificial colonic fluid (ACF) in hours at room temperature (22 °C) or body temperature (37 °C); each group include 6 independent datapoints (n = 6). ·       **Figure 1F**: Speed of algae-MΦNP-robot in artificial colonic fluid (ACF) with TNF-α at body temperature (37 °C); y-axis: motion speed of algae-MΦNP-robot in µm/s, x-axis: the time after transferring algae-MΦNP-robot in artificial colonic fluid (ACF) with TNF-α in hours at body temperature (37 °C); each point includes 100 independent datapoints (n = 100). ·       **Figure 1G**: Speed of algae-MΦNP-robot in artificial colonic fluid (ACF) with IL-6 at body temperature (37 °C); y-axis: motion speed of algae-MΦNP-robot in µm/s, x-axis: the time after transferring algae-MΦNP-robot in artificial colonic fluid (ACF) with IL-6 in hours at body temperature (37 °C); each point includes 100 independent datapoints (n = 100). ·       **Figure 1H**: Speed of algae-MΦNP-robot in artificial colonic fluid (ACF) with IL-1β at body temperature (37 °C); y-axis: motion speed of algae-MΦNP-robot in µm/s, x-axis: the time after transferring algae-MΦNP-robot in artificial colonic fluid (ACF) with IL-1β in hours at body temperature (37 °C); each point includes 100 independent datapoints (n = 100). ·       **Figure 1I**: Speed of algae-MΦNP-robot in artificial colonic fluid (ACF) with IFN-γ at body temperature (37 °C); y-axis: motion speed of algae-MΦNP-robot in µm/s, x-axis: the time after transferring algae-MΦNP-robot in artificial colonic fluid (ACF) with IFN-γ in hours at body temperature (37 °C); each point includes 100 independent datapoints (n = 100). ·       **Figure 1J**: Motile ratio of algae-MΦNP-robot in artificial colonic fluid (ACF) with TNF-α at body temperature (37 °C); y-axis: the motile percentage of algae-MΦNP-robot, x-axis: the time after transferring algae-MΦNP-robot in artificial colonic fluid (ACF) with TNF-α in hours at body temperature (37 °C); each group include 6 independent datapoints (n = 6). ·       **Figure 1K**: Motile ratio of algae-MΦNP-robot in artificial colonic fluid (ACF) with IL-6 at body temperature (37 °C); y-axis: the motile percentage of algae-MΦNP-robot, x-axis: the time after transferring algae-MΦNP-robot in artificial colonic fluid (ACF) with IL-6 in hours at body temperature (37 °C); each group include 6 independent datapoints (n = 6). ·       **Figure 1L**: Motile ratio of algae-MΦNP-robot in artificial colonic fluid (ACF) with IL-1β at body temperature (37 °C); y-axis: the motile percentage of algae-MΦNP-robot, x-axis: the time after transferring algae-MΦNP-robot in artificial colonic fluid (ACF) with IL-1β in hours at body temperature (37 °C); each group include 6 independent datapoints (n = 6). ·       **Figure 1M**: Motile ratio of algae-MΦNP-robot in artificial colonic fluid (ACF) with IFN-γ at body temperature (37 °C); y-axis: the motile percentage of algae-MΦNP-robot, x-axis: the time after transferring algae-MΦNP-robot in artificial colonic fluid (ACF) with IFN-γ in hours at body temperature (37 °C); each group include 6 independent datapoints (n = 6). ·       **Figure 2A**: Quantification of different inputs of MΦNPs binding onto algae through click chemistry; y-axis: MΦNPs binding amount onto algae in mg/ml, x-axis: MΦNPs input amount in mg/ml; each group include 3 independent datapoints (n = 3). ·       **Figure 2C**: Neutralization of TNF-α with different concentration among all groups in vitro; y-axis: remaining TNF-α measured by ELISA in pg/ml, x-axis: equivalent algae amount of each group in × 107/ml; each datapoint include 3 independent studies (n = 3). ·       **Figure 2D**: Neutralization of IL-6 with different concentration among all groups in vitro; y-axis: remaining IL-6 measured by ELISA in pg/ml, x-axis: equivalent algae amount of each group in × 107/ml; each datapoint include 3 independent studies (n = 3). ·       **Figure 2E**: Neutralization of IL-1β with different concentration among all groups in vitro; y-axis: remaining IL-1β measured by ELISA in pg/ml, x-axis: equivalent algae amount of each group in × 107/ml; each datapoint include 3 independent studies (n = 3). ·       **Figure 2F**: Neutralization of IFN-γ with different concentration among all groups in vitro; y-axis: remaining IFN-γ measured by ELISA in pg/ml, x-axis: equivalent algae amount of each group in × 107/ml; each datapoint include 3 independent studies (n = 3). ·       **Figure 2G**: Neutralization of TNF-α with different time among all groups in vitro; y-axis: remaining TNF-α measured by ELISA in pg/ml, x-axis: the time incubating with TNF-α in hours; each datapoint include 3 independent studies (n = 3). ·       **Figure 2H**: Neutralization of IL-6 with different time among all groups in vitro; y-axis: remaining IL-6 measured by ELISA in pg/ml, x-axis: the time incubating with IL-6 in hours; each datapoint include 3 independent studies (n = 3). ·       **Figure 2I**: Neutralization of IL-1β with different time among all groups in vitro; y-axis: remaining IL-1β measured by ELISA in pg/ml, x-axis: the time incubating with IL-1β in hours; each datapoint include 3 independent studies (n = 3). ·       **Figure 2J**: Neutralization of IFN-γ with different time among all groups in vitro; y-axis: remaining IFN-γ measured by ELISA in pg/ml, x-axis: the time incubating with IFN-γ in hours; each datapoint include 3 independent studies (n = 3). ·       **Figure 3C**: Comparison of fluorescence intensity of the capsules prior to oral delivery, in the stomach or small intestine; y-axis: radiant fluorescence of the DiR-dye loaded in the MΦNP PLGA core in Cy7 channel, x-axis: pre-oral delivery, in stomach, and in small intestine groups; each group include 3 independent datapoints (n = 3). ·       **Figure 3E**: Quantified relative fluorescence intensity per gram of colon tissues collected; y-axis: relative fluorescence intensity per gram of tissue, x-axis: the time after oral administration with algae-MΦNP-robot capsule or static algae-MΦNPs capsule; each datapoint include 3 independent studies (n = 3). ·       **Figure 4B & 5B**: Daily mice body weight changes in each group; y-axis: relative bodyweight changes, x-axis: the time after oral administration in days; each datapoint include 6 independent studies (n = 6). ·       **Figure 4C & 5C**: Disease activity index changes in each group; y-axis: disease activity index changes, x-axis: the time after oral administration in days; each datapoint include 6 independent studies (n = 6). ·       **Figure 4D & 5D**: Comparison of the mouse colon length in each experimental group; y-axis: mice colon length in centimeter, x-axis: each group; each group include 6 independent studies (n = 6). ·       **Figure 4E & 5E**: Concentrations of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, and IFN-γ) in the colon tissues; y-axis: the pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, and IFN-γ) amount in nano-gram per gram of colon tissue, x-axis: each group; each group include 6 independent studies (n = 6). ·       **Figure 4G & 5G**: Colonic damage score; y-axis: the colonic damage score, x-axis: each group; each group include 6 independent studies (n = 6). ·       **Figure 4I & 5I**: Colon tissues were excised and analyzed with related mRNA levels of ZO-1; y-axis: relative mRNA levels of ZO-1, x-axis: each group; each group include 6 independent studies (n = 6). ·       **Figure 4J & 5J**: Colon tissues were excised and analyzed with related mRNA levels of Occludin; y-axis: relative mRNA levels of Occludin, x-axis: each group; each group include 6 independent studies (n = 6). ·       **Figure 6B**: Daily mice body weight changes in each group; y-axis: relative bodyweight changes, x-axis: the time after oral administration in days; each datapoint include 6 independent studies (n = 6). ·       **Figure 6C**: Comprehensive blood chemistry panel; y-axis: relative to normal level of each blood chemistry panel, x-axis: ALB, albumin; ALP, alkaline phosphatase; ALT, alanine transaminase; AMY, amylase; TBIL, total bilirubin; BUN, blood urea nitrogen; CA, calcium; PHOS, phosphorus; CRE, creatinine; GLU, glucose; Na+, sodium; K+, potassium; TP, total protein; GLOB, globulin; each group include 3 independent studies (n = 3). ·       **Figure 6D**: Counts of various blood cells; y-axis: cell counts in number/µl, x-axis: WBC, white blood cells; RBC, red blood cells; PLT, platelets; each group include 3 independent studies (n = 3). ·       **Figure 6G**: Concentrations of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, and IFN-γ) in the health colon tissues; y-axis: the pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, and IFN-γ) amount in nano-gram per gram of colon tissue, x-axis: PBS, mice oral administration with 1X PBS as health control; algae-MΦNP-robot capsule, mice oral administration with algae-MΦNP-robot capsule; each group include 3 independent studies (n = 3). **Data statistical analysis:** The experiments conducted were all performed repeatedly and independently to acquire the data shown in figures, which were reported as error bars represent ± standard deviation relative to means. To assess the statistical significance between two groups, a two-tailed, Student’s *t* test was used, and for multiple comparisons, a one-way analysis of variance (ANOVA) with Dunnett’s test was performed. Statistical significance is denoted as **P* &lt; 0.05, ***P* &lt; 0.01, ****P* &lt; 0.001, and *****P* &lt; 0.0001. To minimize any potential bias that might occur during the study, no data were excluded; samples were allocated randomly to different experimental groups; organisms were cultured under the same environmental conditions and randomly allocated to each group; investigators were not blinded during data collection and analysis throughout the experiments. ## Sharing/Access information We don't have any other sharing/access information about the data. ## Code/Software We don't have any code or software in the manuscript.