The swarmable magnetic-driven nanorobots for facilitating trans- intestinal mucosal delivery of oral vaccines to enhance mucosal and systemic immune responses

The swarmable magnetic-driven nanorobots for facilitating trans- intestinal mucosal delivery of oral vaccines to enhance mucosal and systemic immune responses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The swarmable magnetic-driven nanorobots for facilitating trans- intestinal mucosal delivery of oral vaccines to enhance mucosal and systemic immune responses Linghong Huang, Xinyuan Sun, Quan Zhang, Jun Long, Xuewu Chen, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5388438/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The continuous secretion of mucus by the intestinal mucosa and the intestinal motility combine to limit the absorption of orally administered vaccines. To extend the residence time of vaccines within the gastrointestinal tract and to improve their mucosal transit, we have developed a technology capable of swiftly and actively traversing the intestinal mucus barrier. In this study, we synthesized a biodegradable magnetic driven nanorobot (MNC@CaMn) loaded with antigen and constructed a magnetic driven nanorobot vaccine delivery platform. Under the precise regulation of the magnetic field, the residence time of the vaccines in the intestine was significantly prolonged, and the vaccine exhibited a swarming motility that could rapidly converge and cross the intestinal mucus barrier in a targeted manner, thus greatly facilitating antigen delivery and presentation and significantly activating CD8 + T lymphocytes. In addition, the rough surface of the nanorobot ensured stable antigen loading, while the Mn 2+ in the particles was able to stimulate efficient mucosal and systemic immune responses due to its excellent adjuvant effect. The magnetic driven nanorobot vaccine delivery system constructed in this study provides a new strategy for the development of efficient oral and mucosal vaccines. mucosal immunization oral vaccine magnetic driven nanorobot swarming motility Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Given that the gut-associated lymphoid tissue (GALT), the body's largest immune center, houses approximately 70% of all immune cells, oral vaccines have emerged as a cutting-edge strategy for triggering a strong immune response[ 1 ]. Compared with the traditional intramuscular or subcutaneous routes of vaccination, oral vaccines activate both mucosal and systemic immune responses[ 2 – 5 ], offering advantages such as simplicity of administration, improved patient acceptance, and cost-effectiveness[ 6 ]. Polio and rotavirus vaccines, which are commercially available today, are successful examples of oral vaccines. However, the promotion of oral vaccines is constrained by the harsh environment of the gastrointestinal tract, specifically, two major challenges: first, the destruction of vaccine activity by gastric acid and digestive enzymes; and second, the continuous secretion of mucus from the intestinal mucosa forms a physical barrier, which, together with intestinal motility, restricts the effective absorption of vaccine components[ 7 ]. Advances in enteric capsule technology have effectively overcome the threat of gastric acid and digestive enzymes to vaccines, but the intestinal mucus layer remains a significant barrier to the efficiency of oral vaccine absorption. Innovative delivery technologies are revolutionizing the way oral vaccines cross the mucus barrier. Targeting the hydrophobic and negatively charged properties of mucin in mucus, DNA vaccines have been delivered using cationic viscous polymers to improve their retention in the gut and enhance antitumor immune responses[ 8 ]. However, the hydrophobic cationic polymers bind excessively to the mucus, which in turn impedes the normal diffusion of the antigen[ 9 ]. To address this challenge, Zhang et al. [ 3 ] utilized hydrophilic and slightly negatively charged “mucus-inert” polyethylene glycol (PEG) derivatives to encapsulate antigens and cell-penetrating peptides. In the mucus environment, the PEG coating dissociates in time, exposing a core rich in cell-penetrating peptides, which facilitates the smooth penetration of the antigen into the epithelial cells and enhances its bioavailability. Although these innovative strategies help to improve the ability of the vaccine to cross the mucus, they are all passive and difficult to overcome the strong resistance of the mucus layer. In the future, we will focus on the development of more efficient and active mucus-crossing technologies, aiming to enable oral vaccines to cross the mucus layer rapidly and precisely, and to enhance the efficiency of the immune response of oral vaccines. Magnetic-driven technology has a growing potential in the biomedical field, enabling precise manipulation in a non-invasive manner, and effectively crossing biological tissue barriers[ 10 , 11 ]. Magnetic-driven nanorobots, with their tiny size, excellent autonomous mobility, and precise navigation performance, make them a preferred choice for drug delivery. For tissues such as the gastrointestinal tract, which has a large cavity, magnetic-driven nanorobots can easily realize all-around autonomous movement under the guidance of a local magnetic field, which opens up new possibilities for efficiently breaking through the mucus layer and accelerating drug delivery. Although the motion characteristics, functionalization, and manipulation of individual magnetic micro-nanorobots have been deeply explored, how to precisely manipulate the swarms of nanorobots to achieve on-demand drug and energy delivery is still a technical challenge to overcome[ 12 ]. Compared to single particles, the advantage of population control is that it significantly improves the dose and efficiency of drug delivery, while enhancing the image contrast for drug tracking and monitoring, providing a better solution for drug delivery visualization. Unfortunately, there is a scarcity of dedicated research on oral magnetic driven delivery systems. Zhang et al. [ 13 ] used magnetically controlled microneedles guided by an external magnetic field to puncture the intestinal mucosa and successfully deliver insulin. However, direct puncture of the intestinal mucosa by microneedles may increase the risk of infection. Given this, the development of novel magnetic-driven nanorobots as vaccine carriers is expected to improve the efficiency of vaccines across the mucus barrier while avoiding the potential risk of infection. To overcome the mucus barrier and gastrointestinal peristalsis, and prolong the retention time of antigens in the intestine, we focused on the development of a technology capable of rapidly and actively crossing the intestinal mucus layer. In the present study, we prepared biodegradable magnetic-driven nanorobots (MNC@CaMn) loaded with antigen and constructed a magnetic-driven nanorobot vaccine. Under the precise regulation of an external magnetic field, this vaccine was able to exhibit a collective behavior similar to that of a swarm of bees, which rapidly assembled and oriented to traverse the intestinal mucus layer, significantly prolonging the retention time of antigens in the intestines, which in turn facilitated the effective delivery and presentation of antigens. In addition, the rough texture of the surface of the nanorobot ensured the stable loading of antigens, and the Mn 2+ contained in the particles acted as an adjuvant to activate the antigen-presenting cells in the spleen and mesenteric lymph nodes, thus realizing the dual activation of both efficient mucosal immunity and systemic immunity (Schema 1). This work provides a new perspective for the development of magnetic-driven nanorobots for oral and mucosal vaccine delivery systems. 2 Methods 2.1 Materials CaCl 2 , Na 2 CO 3 , FeCl 3 , MnCl 2 ·H 2 O, and potassium citrate were purchased from Macklin (China). Mucin powder was purchased from Yuanye Bio-Technology Co., Ltd (China). Ovalbumin (OVA) was purchased from Sigma (USA). BCA kit, cell counting kit-8 (CCK8), Lyso-Tracker and DAPI were purchased from Beyotime Biotechnology (China), Ovalbumin antibody was purchased from LifeSpan BioSciences (US). Anti-IgG, anti-IgG1, anti-IgG2a and anti-IgA antibody were purchased Abcam (UK). The CD11C antibody, CD68 antibody and CD8-GB13429 were purchased from Servicebio (China). All flow cytometry antibody dyes and cytokine ELISA detection kits were purchased from BioLegend (USA). All female C57BL/6 mice (4–6 weeks) used in the study were purchased from Beijing HFK Laboratory Animal Technology Co. (China). In addition, all animal experiments were eligible and approved by the Ethics Committee of Jinan University. 2.2 Preparation and characterization of MNC@CaMn nanorobots Preparation of magnetic nanoclusters (MNCs): First, potassium citrate (0.4 g) and FeCl 3 (0.65 g) were dissolved in ethylene glycol (40 mL) and vigorously stirred at room temperature for 1 h. Then, sodium acetate (1.2 g) was added and stirred for 30 min. The mixture was then transferred to a high-pressure reaction kettle and reacted at 200°C for 12 h. Finally, the product was collected, washed three times with ethanol and water, and stored by freeze-drying. Preparation and characterization of MNC@CaMn nanorobots: First, CaCl 2 and MnCl 2 mixed solution (0.016 M, molar ratio of Ca 2+ /Mn 2+ = 1:1) was prepared using glycerol/water solution (1/1, v/v). Then, MNCs (5 mg) were added to the CaCl 2 and MnCl 2 mixed solution (10 mL) and stirred for 30 min. then NH 4 HCO 3 (10 mL, 0.16 M) in glycerol/water solution (1/1, v/v) was added to the mixture and stirred for 1 h at 50°C. and the precipitate was centrifuged and washed three times. Finally, the formed nanoparticles were characterized by scanning electron microscopy (SEM, Zeiss, Germany), transmission electron microscopy (TEM, JEM-2010HR, Japan), X-ray powder diffractometer (XRD, Rigaku, Japan), Fourier infrared spectroscopy (FT-IR, VERTEX70, Germany), laser nanoparticle sizer (Malvern, Britain) and vibrating sample magnetometer (VSM) (LakeShore, US). In addition, 150 mg of mucin was taken in 5 mL of deionized water to prepare artificial intestinal mucus. Then, 5 mg of magnetic nanorobots were added to this intestinal simulated mucosal fluid. The ability of the magnetic nanorobots to penetrate the mucus layer in the presence or absence of a magnetic field was observed and recorded. 2.3 The loading of OVA on MNC@CaMn nanorobots First, 1 mL of OVA solution (1 mg/mL) was prepared using saline. Then, the MNC@CaMn nanorobots (5 mg) were added to OVA solution (1 mL), and the suspension was stirred at room temperature. The free OVA in the solution was detected at different times using a BCA kit. 2.4 Swarm motion detection by MNC@CaMn nanorobots First, the labyrinth was modeled with transparent resin by using 3D printing technology, and then water, DMEM, or artificial mucus was added to the labyrinth. Subsequently, a suspension of MNC@CaMn nanorobots was dropped into the labyrinth. Next, a rotating permanent magnet was placed underneath the labyrinth to aggregate the MNC@CaMn nanorobots, and then the rotating magnet was moved to move the aggregated FMP through the labyrinth. 2.5 Co-localization of magnetic-driven nanorobots in DCs First, the hind limb bones were isolated from female C57BL/6 mice. Then, the two distal bone ends were excised, and the bone marrow cells were flushed with RPMI 1640. The red blood cells in bone marrow cells were lysed and the remaining cells were centrifuged at 1500 rpm for 10 min. Subsequently, the cells were seeded into 6-well plates and cultured in RPMI 1640 medium (containing 20 ng/mL GM-CSF and 10 ng/mL IL-4) for 6 days. The medium was changed every two days. On day 6, immature BMDCs were obtained. Next, DC2.4 and BMDCs (5 × 10 4 cells/dish) were inoculated in cell culture dishes. After incubation, the cells were incubated with Cy5.5-OVA or MNC@CaMn/Cy5.5-OVA preparations for 6 h. Subsequently, the cells were incubated with Lyso-Green fluorescent dye for 2 h and fixed for 20 min, and then stained with DAPI dye solution for 5 min. Finally, the cells were observed with a confocal laser scanning microscope (CLSM, ZEISS, Germany). 2.6 Stimulation of BMDCs and BMDMs in vitro The above immature BMDCs were inoculated into 24-well low-attachment surface plates (2×10 5 cells/well) and treated with PBS, OVA, MNC@CaMn/OVA, and LPS/OVA solution (OVA: 5 µg/well) for 24 h. Then, the fluorescent dye-labeled antibodies solutions (anti-CD11c-APC, anti-CD86-PerCP-Cy5.5, anti-CD80-PerCP-Cy5.5 and anti-H2Kb/SIINFEKL (MHC I)-PE) to detect DC maturity and the expression of OVA-specific MHC I with a flow cytometer (Beckman Coulter, USA). 2.7 In vivo evaluations of oral vaccine levels in the gastrointestinal tract and mesenteric lymph nodes First, MNC@CaMn nanorobots loaded with Cy5.5-OVA were used to prepare fluorescent magnetic-driven nanorobot vaccines. Subsequently, these nanorobot vaccines were administered by gavage to C57BL/6 female mice (4–6 weeks old). Then, the mice were randomly divided into two groups (n = 4), one group of mice was magnetically treated and the other group was not magnetically treated (100 µg Cy5.5-OVA/mouse, 1 mg MNC@CaMn/mouse). The fluorescence intensity in the gastrointestinal tract area of mice was detected with a small animal bioluminescence imaging system (IVIS Lumina III, PerkinElmer, USA) at 1, 6 and 24 h after gavage. In addition, at 6 and 24 h after gavage, the mouse mesenteric lymph nodes and gastrointestinal tract tissues were collected and their fluorescence intensity were examined with the small animal bioluminescence imaging system. All of the above mice were starved overnight before gavage, and 100 µL of 1% NaHCO 3 solution was administered orally 30 min before gavage to neutralize gastric acid. 2.8 In vivo immunization assessment of oral vaccines C57BL/6 female mice (4–6 weeks old) were randomly divided into 5 groups (n = 5) and gavaged with 100 µL of OVA, MNC@CaMn/OVA, MNC@CaMn/OVA (M), and Alum/OVA solution (OVA: 100 µg/mouse). The mice were vaccinated four with interval of 4 days. Then, 0.05 g of fresh feces were collected from mice on days 0, 8, 12 and 16 and made into a suspension of 0.1 g/mL. Subsequently, the suspension was centrifuged at 12,000 rpm for 30 min to collect supernatant. On the 4th day after the last inoculation, serum was separated from the blood of the experimental mice and stored at -20℃. Subsequently, the splenocytes and lymphocytes were isolated from collected spleens and mesenteric lymph nodes of mice. All of the above mice were starved overnight before gavage. The mice were gavaged with 100 µL of 1% NaHCO 3 solution to neutralize the gastric acid 30 min prior to gavage of the nanorobot. Detection of the ability of nanorobots to pass through the intestinal mucosa: the mouse intestines were removed, fixed with paraformaldehyde, paraffin-embedded, sectioned and immunohistochemically stained, and the mouse intestines were observed by a fluorescence microscope (DMRA2, Leica, Germany). DC and macrophage recruitment and migration assays: the mouse ileal tissues and peyer patchs were collected, fixed with 4% paraformaldehyde, sectioned, and stained by immunofluorescence using CD11C and CD68 antibody dyes. Then, the recruitment of DCs and macrophages in the mouse ileum and their migration to the peyer patchs were observed with the fluorescence microscope. Immunohistochemical analysis of peyer patchs and mesenteric lymph nodes: the mouse ileal tissues, peyer patchs, and mesenteric lymph nodes were collected and fixed with 4% paraformaldehyde for immunohistochemical analysis. The distribution of antigen protein OVA in the ileal tissues, peyer patchs, and mesenteric lymph nodes was observed with the fluorescence microscope. Detection of serum and mucosal antibody titers: the enzyme-linked immunosorbent assay (ELISA) technique was used to detect the levels of OVA-specific antibodies (IgG, IgG1 and IgG2a) in the serum of experimental mice as well as the levels of IgA antibodies in the fecal supernatant. Detection of cytokines secreted by splenocytes: the obtained splenocytes were inoculated into 12-well plates (5×10 5 cells/well) and then stimulated with antigen OVA solution (final concentration: 25 µg/mL) for 60 h. Subsequently, the cellular supernatants were collected to detect the secretion levels of cytokines IFN-γ, TNF-α, IL-6, and IL-4 by using an ELISA kit. Detection of T-cell ratio in splenocytes and lymphocytes: the Splenocytes and lymphocytes (1×10 6 cells/mouse) were extracted from the above mice and stained with fluorescent dye-labeled antibodies (anti-CD3-APC, anti-CD8α-PerCP-Cy5.5 and anti-CD4-FITC). Subsequently, the cells were washed with PBS and resuspended. Finally, the cells were detected with the flow cytometer. Detection of immune memory T-cell ratio: the Splenocytes and lymphocytes (1×10 6 cells/mouse) were extracted from the above mice and stained with fluorescent dye-labeled antibodies (anti-CD62L-APC, anti-CD44-PE, anti-CD8α-PerCP-Cy5.5 and anti-CD4-FITC). Subsequently, the cells were washed with PBS and resuspended. Finally, the cells were detected with the flow cytometer. Evaluation of histocompatibility of vaccine formulations: the Hearts, livers, spleens, lungs, stomachs, ileums and kidneys of these experimental mice were collected and fixed with 4% paraformaldehyde. Subsequently, tissue sections were made from these organs and stained with hematoxylin-eosin (H&E) staining solution. Finally, the fluorescence microscope was used to observe the sections. 2.9 In vivo antitumor immunological evaluation of oral vaccines C57BL/6 female mice (4–6 weeks old) were randomized into 5 groups (n = 5). On day 0, B16-OVA cells were injected subcutaneously into the left dorsum of each mouse. On days 7, 11, and 15, mice were orally inoculated with 100 µL of Saline, OVA, MNC@CaMn/OVA, or MNC@CaMn/OVA (M) formulations, and their body weights and tumor volumes were monitored every 1 day. On day 19 after tumor inoculation, the spleens of mice were collected and the lymph nodes and tumors were drained. First, the collected tumors were sectioned and stained with CD8 + T cell staining solution, and the infiltration of CD8 + T cells in the mouse tumors was observed by the fluorescence microscope. Subsequently, splenocytes (1×10 6 cells/one) were collected from the spleen, stained with APC-anti-CD3, Percp-Cy5.5-anti-CD8a and FITC-anti-CD4, and detected by the flow cytometry. 2.10 Statistical analysis GraphPad Prism 5 and Origin software were used to statistically analyze the experimental results, and one-way ANOVA test was used to analyze the differences between the experimental groups. The experimental data were expressed as Mean ± SEM. Where *p < 0.05 , **p < 0.01 and ***p < 0.001 were used to indicate significant differences. 3 Results and Discussion 3.1 Preparation and characterization of magnetic-driven nanorobots First, magnetic nanoclusters (MNCs) were prepared by hydrothermal method. Subsequently, calcium-doped manganese carbonate was further deposited on the MNC surface with Mn 2+ , Ca 2+ and sodium bicarbonate to obtain MNC@CaMn nanorobots with core-shell structure. This synthesis strategy is a green chemistry reaction. The SEM images demonstrated that the MNCs had a spherical morphology (Fig. 1 a), whereas the MNC@CaMn nanoparticles displayed an ellipsoidal morphology with sizes of approximately 200 nm and 600 nm, respectively. (Figs. 1 b&c). The elemental analysis (Fig. 1 d) confirmed the presence of calcium (Ca), manganese (Mn), carbon (C) and oxygen (O) in the nanorobots. Subsequent XRD patterns (Fig. 1 e) showed that the diffraction peaks of MNC@CaMn nanorobots at 2θ = 35.18°, 42.61°, 56.68°, and 62.40° corresponded to the (311), (400), (511), and (440) crystal planes of MNC nanoparticles in the standard XRD patterns[ 14 ], respectively; while the diffraction peaks at 2θ = 23.31°, 29.92°, 40.11°, and 49.29° correspond to the (012), (104), (113), and (116) crystal planes of MnCO 3 in the standard XRD patterns. All these results indicate the successful preparation of MNC@CaMn nanorobots. Next, nanorobot-loaded model antigen OVA was used to evaluate its antigen loading efficiency. The infrared spectrum (IR) was shown in Fig. 1 f, the IR spectrum of MNC@CaMn showed stretching vibration absorption peaks of Fe-O-Fe near 581 cm − 1 [ 15 ]and bending vibration absorption peaks of CO 3 2− near 869 cm − 1 [ 16 ]. While the IR spectrum of MNC@CaMn/OVA exhibit a new characteristic peak near 1650 cm − 1 upon adsorption of protein antigen, primarily attributed to the stretching vibration of C = O in the amide group of the protein[ 17 ]. Broad peaks corresponding to N-H stretching vibrations were observed in the range of 3000–3750 cm − 1 , while peaks associated with C-H stretching vibrations were detected in the range of 2700–3000 cm − 1 [ 18 ]. This indicates that the MNC@CaMn nanorobots can effectively adsorb antigen OVA. Additionally, the BCA assay was employed to quantify the antigen loading capacity of MNC@CaMn nanorobots at different time points, as shown in Fig. 1 g. The antigen loading capacity of MNC@CaMn nanorobots gradually increased over time, reaching maximum adsorption capacity (approximately 200 µg/mg) within 12 h and maintaining stable loading over an extended period. This will help in co-delivery of antigen and adjuvant. Furthermore, the XPS full spectrum of the MNC@CaMn/OVA nanorobots (Fig. 1 h) revealed characteristic binding energy peaks for N 1s confirming the presence of N elements in the MNC@CaMn/OVA nanorobots. Elemental spectra (Figs. 1 i) further supported these findings, with peaks at 399.7 and 400.3 eV corresponding to the N 1s of OVA, indicating that MNC@CaMn nanorobots can effectively adsorb protein antigen. In addition, the vibrating sample magnetometer (VSM) and an NdFeB magnet block were used to examine the magnetic properties of MNC@CaMn nanorobots. As shown in the hysteresis line in Fig. 1 j, both MNC and MNC@CaMn nanorobots exhibit superparamagnetism with coercivity close to zero. Their saturation magnetization strengths were 18.40 and 65.56 emu/g, respectively. Subsequently, the dispersed MNC@CaMn nanorobots rapidly aggregated when the NdFeB magnet was attached to the vial wall and completely aggregated within 10 s (Fig. 1 k). These results indicate that the MNC@CaMn nanorobots maintain good magnetic properties. Given that this project plans to use magnetic-driven nanorobots loaded with antigens for trans-intestinal mucus transport, we prepared artificial mucus using mucin to evaluate the trans-mucus ability of magnetic-driven nanorobots in vitro. As shown in Fig. 1 l, the artificial mucus was relatively viscous and the nanorobots could not effectively traverse the artificial mucus without magnet guidance. However, with magnet guidance, the nanorobots could rapidly traverse the artificial mucus, suggesting that the magnetic-driven nanorobots can effectively cross the mucus layer. 3.2 Navigational locomotion of nanorobots Magnetic driven and control technology has attracted much attention due to its remarkable potential for biomedical applications[ 19 ]. The advantage of this technology lies in the ability of the magnetic field to penetrate deep into biological tissues with virtually no side effects. For performing specific tasks in the complex intestinal environment, nanorobots must be highly intelligent in order to work effectively in the intestine. Compared to a single magnetic-driven nanorobot, swarm motions of nanorobots show greater advantages in achieving efficient drug delivery. In this study, we designed a complex labyrinth experiment to simulate the navigational ability of MNC@CaMn nanorobots in the intestine. The labyrinth is made of transparent resin material and fabricated by 3D printing technology. In the experiment, we used a rotating magnetic field to drive the nanorobots to demonstrate its clustering effect. Driven by the rotating magnetic field, the nanorobots rapidly aggregated to form a microcolony like a swarm of bees, and successfully completed a variety of continuous motions including straight-line advancement and turning (Fig. 2 a and Supporting Information Video S1). In addition, the nanorobots also demonstrated its excellent navigation performance in a channel containing DMEM cell culture medium and artificial mucus (Fig. 2 b&c and Supporting Information Videos S2&S3). These experimental results show that the nanorobots can efficiently traverse the mucus layer and realize the intended movement path even in a tiny and structurally complex environment. Further observations revealed that in the zoomed-in video (Fig. 2 d and Supporting Information Video S4), multiple hydrodynamic vortices are generated around the nanorobots when a rotating magnetic field is applied. There are interactions between these vortices that cause the nanorobots to be attracted to each other, thus causing the entire cluster to narrow down and all the nanorobots to be dynamically confined to a smaller area like a whole. This phenomenon not only facilitates the control of the cluster of nanorobots, but also simplifies the process of transporting them to the target location. Effective manipulation and precise positioning of the nanorobots can be achieved through simple magnetic field adjustments. 3.3 Nanorobots facilitate antigen delivery to DCs Antigen uptake by antigen-presenting cells (APCs) is a critical first step in initiating antigen-specific immune responses[ 20 ]. DCs, as the most specialized APCs, capture exogenous antigens, up-regulate co-stimulatory molecules to promote DCs maturation and migrate to lymph and spleen to deliver antigens to T cells via MHC molecules and activate T cells[ 21 ]. To investigate the distribution of MNC@CaMn nanorobots loaded with antigen in APCs and their effects on cellular functions, we employed confocal laser scanning microscopy (CLSM) to observe the distribution of nanorobots loaded with Cy5.5-labeled OVA (Cy5.5-OVA) in DC2.4 and BMDCs. As shown in Fig. 3 a-d, Cy5.5-OVA alone entered the lysosomes of DC2.4 and BMDCs in relatively small amounts, while the nanorobots loaded with Cy5.5-OVA significantly increased the overlap of red fluorescence of antigens with the green fluorescence of lysosomes in DC2.4 and BMDCs. This result suggests that the nanorobots significantly increases the uptake efficiency of antigen by DCs. In addition, we noted that in the antigen alone group, BMDCs presented a typical rounded appearance, indicating that they were immature. Whereas, after stimulation with the nanorobots, BMDCs transformed into a polypod-like morphology (red arrows in Fig. 3 c), which suggests that BMDCs were stimulated to mature. Together, these results confirm that the antigen loaded by the nanorobots significantly enhances the uptake of antigen by DCs and promotes the maturation of DCs, thus providing favorable conditions for the subsequent immune response. 3.4 Nanorobot vaccines promote BMDC maturation and antigen presentation and stimulate BMDM polarization towards M1-type macrophages APCs are the cells in the organism that have the ability to uptake, process and present antigen information and induce immune responses in T and B cells. They mainly include dendritic cells and macrophages. The high expression of exogenous antigenic peptide-specific MHC class I molecules in DCs implies that antigens are cross-presented, which is essential for activating anti-tumor immunity[ 22 ]. In addition, macrophages are similarly activated by antigen and differentiate into M1-type and M2-type macrophages. M1-type macrophages have strong tumor-killing and antigen-presenting abilities, which can enhance anti-tumor effects by activating anti-tumor T-cell responses. Therefore, reversal of immunosuppressive M2-type macrophages to M1-type macrophages is an important strategy to enhance anti-tumor immunotherapy[ 23 ]. In this study, isolated BMDCs were treated with MNC@CaMn nanorobot vaccines for 24 h and analyzed for the expression of OVA-specific MHC I, MHC II, CD80 and CD86 molecules. As shown in Fig. 4 a and Figs. 4 c&d, OVA alone only slightly increased CD80/CD86 co-expression and hardly increased MHC I expression, indicating that OVA alone did not significantly promote BMDC maturation and MHC I-mediated antigen presentation. In contrast, antigen loading by the nanorobots significantly increased CD80/CD86 co-expression and MHC I expression, suggesting that the nanorobot vaccines significantly BMDC maturation and MHC I-mediated antigen presentation. This was attributed to the role of Mn 2+ in the nanorobots that could significantly increase cellular immune responses. Subsequently, after macrophages BMDMs were extracted from mouse bone marrow and induced to differentiate into M2-type macrophages using IL4, the proportion of M2-type macrophages that were reversed to M1-type macrophages was analyzed by treating with the nanorobot vaccines for 24 h. The results were shown in Figs. 4 b&e, CD206 molecules were significantly down-regulated while CD86 molecules were significantly up-regulated in the nanorobot vaccines group compared to the NC and OVA groups. Its CD86/CD206 ratio was close to that of the positive control LPS, suggesting that the nanorobots could significantly promote the reversal of M2-type macrophages to M1-type macrophages, which would significantly enhance the anti-tumor immune response. 3.5 Magnetic fields promote retention of nanorobot vaccines in intestinal tissues and migration to mesenteric lymph nodes Longer retention of antigen at the inoculation site and migration to peripheral immune organs can promote sustained immune stimulation and thus enhance the immune response. Here, the role of magnetic-driven nanorobots on antigen retention and migration in gastrointestinal tissues was investigated. As shown in Figs. 5 a&b, at 6 h after immunization, the fluorescence in the abdomen of mice with nanorobot without magnetic field treatment decreased rapidly, and only weak fluorescence could be observed at 24 h. However, the fluorescence in the abdomen of mice showed a stronger fluorescence signal when the nanorobot vaccines was treated with a magnetic field for 6 h. This is due to the fact that the originally dispersed nanorobots in the gastrointestinal tissues were aggregated by the magnetic field and concentrated to the abdominal surface of the mouse gastrointestinal tract, resulting in the detection of a stronger fluorescence signal. Moreover, when the magnetic field was withdrawn, the mouse inoculation site still contained a large amount of fluorescent signals at the 24 h. This suggests that the antigen-loaded MNC/CaMn nanorobots can achieve longer antigen retention time in the gastrointestinal tissues under magnetic field. At 6 h and 24 h after immunization, the gastrointestinal tissues and mesenteric lymph nodes of mice were isolated to further detect the retention and migration of OVA. The gastrointestinal tissues are shown in Figs. 5 c-e, and the OVA content in the gastrointestinal tract was significantly higher at 24 h in the magnetic field-treated group, compared with the no magnetic field group. The OVA content in the gastrointestinal tract was significantly higher in the magnetic field-treated group. This future indicates that the magnetic field prolonged the residence time of antigen in the gastrointestinal tissues. Subsequently, the results of mesenteric lymph nodes were shown in Figs. 5 f&g. At both 6 h and 24 h, the OVA content in the mesenteric lymph nodes of the group with magnetic field was higher than that of the group without magnetic field. This suggests that the magnetic field increases the retention time of the nanorobot vaccine at the vaccination site and also helps to promote antigen migration to the mesenteric lymph nodes, which contributes to the immune response. 3.6 Magnetic-driven nanorobots cross the mucus layer and recruit APCs to increase mucosal immune response In this study, magnetic-driven nanorobots were orally administered to the mice, and a vortex magnetic field was applied to their abdomens to test whether the nanorobots could successfully traverse the intestinal fluid and stimulate both mucosal and systemic immune responses. The vaccine administration schematic is shown in Fig. 6 a. Antibody titer is a key indicator of the level of immune response. Mucosal vaccination induces not only systemic IgG antibodies, but also secretion of IgA antibodies from the mucosal surface[ 24 , 25 ]. Therefore, the levels of OVA-specific IgG, IgG1 and IgG2a antibodies in the serum of immunized mice and IgA antibodies in fresh feces were detected by ELISA. As shown in Figs. 6 b-d, the Alum/OVA group did not significantly increase the levels of systemic IgG, IgG1 and IgG2a antibodies compared to the OVA alone group, suggesting that it is difficult to augment the immune response to an oral vaccine using only a simple aluminum adjuvant. In addition, the loading of OVA by the MNC/CaMn nanorobots only significantly increased the serum levels of IgG1 antibodies and did not significantly enhance the serum levels of IgG and IgG2a antibodies compared to the OVA alone group. However, nanorobots with magnetic fields significantly increased the expression of antigen-specific antibodies IgG and IgG2a compared to nanorobots without magnetic fields. This suggests that that magnetic nanorobots significantly increase the immune response, especially the cellular immune response, in the presence of a magnetic field. Moreover, the results of IgA antibody detection in fresh feces are shown in Fig. 6 g-k. After immunization with OVA alone, the level of IgA antibody increases rapidly, then decreases sharply after the 8th day, and it cannot be maintained for a long period of time to induce the production of a high level of IgA antibody, and the MNC/CaMn/OVA nanorobot vaccines without magnetic field also didn’t effectively increase the level of IgA antibody. In contrast, the group of MNC/CaMn/OVA nanorobot vaccines with magnetic field showed a rapid increase in IgA antibody levels within 8 d and did not decrease significantly on both the 12th and 16th d. This suggests that oral administration of the MNC/CaMn/OVA nanorobot vaccines alone is not sufficient to increase the level of mucosal immune response to the antigens, which may be attributed to the fact that, in the absence of a magnetic field, the magnetic-driven nanorobot vaccines cannot traverse the mucosal layer of the gastrointestinal tract and cannot be uptaken by the immune cells in the epithelium of the gastrointestinal tract, whereas application of a magnetic field induces the magnetic-driven nanorobot vaccines to traverse the mucosal layer actively and rapidly, and prolongs their residence times in the gastrointestinal tract, which will be conducive to the production of mucosal and systemic antibodies. The small intestine is the target site for all currently approved oral vaccines and most studies reported[ 6 ]. Unlike the lining of the oral and gastric cavities, the intestinal mucosa recognizes foreign antigens and transports them through the mucosal barrier to the immune system[ 26 ]. Therefore, the oral vaccine in this research mainly chooses the small intestine as the object of analysis. Nanorobot vaccines passing through a magnetic field can increase the retention of antigens at the inoculation site. However, whether the nanorobot successfully carries antigens across the intestinal mucus layer in the presence of a magnetic field still requires further testing. Next, immunohistochemical analysis was performed to further detect the level of antigen crossing the intestinal mucus layer. The results are shown in Fig. 6 e, OVA-positive areas (brown areas) were rarely observed in the OVA-alone group, whereas a large number of OVA-positive regions appeared in the intestinal mucus after the antigen-loaded MNC/CaMn nanorobots were treated with a magnetic field, and could be detected in the submucosal layer in the intestinal mucosa. This further suggests that the combined use of magnetic field and magnetic-driven nanorobots helps to prolong the residence time of the antigen in the intestine and promotes the penetration of the antigen into the intestinal mucosal layer, which would contribute to the uptake of the antigen by the APCs. The foundation of mucosal immunity is that APCs recruit and capture antigens at mucosal inductive sites (such as PPs), and then migrate through lymphatics to local mucosal-draining lymph nodes (such as mesenteric lymph nodes). Here, APCs present antigens to T cells, initiating both mucosal and systemic immune responses, while also generating antigen-specific memory cells and antibody-secreting cells, thereby establishing a long-lasting effective immune response against specific antigens[ 27 ]. Therefore, the successful recruitment of immune cells at the mucosal induction site is crucial for the mucosal immune response. We first observed the recruitment of APCs throughout the intestinal mucosal site, and the results, as shown by immunofluorescence staining images in Fig. 6 f, showed that the OVA alone and Alum/OVA groups recruited fewer DCs and macrophages, while the MNC/CaMn/OVA(M) group recruited more DCs and macrophages. It may be due to the fact that, the magnetic field effectively increased the retention and transmucosal layer efficiency of the magnetic-driven nanorobots loaded with antigen at the inoculation site, stimulated the intestinal epithelial tissue, and recruited more APCs. Next, APCs recruited in local mucosal-draining lymph nodes (such as PPs) were examined. The results were shown in Fig. 7 a, there were fewer DCs and macrophages in the PPs in the OVA alone and Alum/OVA groups, while there were significantly more DCs and macrophages in the PPs of the MNC@CaMn/OVA (M) group, which may be attributed to the fact that MNC@CaMn/OVA can rapidly aggregate and rapidly cross the intestinal mucus layer under magnetic field, stimulating the intestinal mucosa and recruiting more DCs and macrophages. This would help APCs to take up antigen and be stimulated to mature and accelerate migration into mesenteric lymph nodes. 3.7 The migration of APCs to mesenteric lymph nodes promotes antigen presentation After uptake of antigens, APCs need to migrate to lymph nodes for presenting antigens to T lymphocytes, which trigger an immune response. Although macrophages in APCs can also present antigens, their main functions are phagocytosis and antigen presentation in tissues, rather than migrating to immune organs to activate T cells[ 28 ]. Therefore, it is mainly activated DCs in APCs that have the function of migrating to secondary lymphoid organs and activating T cells[ 29 ]. Therefore, DCs in mesenteric lymph nodes, an important secondary lymphoid organ in mucosal immunity to oral vaccines, were examined and the results are shown in Fig. 7 b. There were significantly more DCs in the mesenteric lymph nodes in the MNC@CaMn/OVA (M) group compared to the OVA alone and Alum/OVA groups, suggesting that activated DCs migrated to the mesenteric lymph nodes in greater quantities, which is essential for the activation of mucosal immune responses by oral vaccines. Next, immunohistochemical analyses were performed to further examine the level of antigen being transported to the mesenteric lymph nodes after uptake by APCs. First, the level of antigen enrichment in mucosal inductive sites PPs was examined. As shown in Fig. 7 c, inoculation with OVA alone resulted in less OVA-positive areas (brown areas) in PPs, whereas MNC@CaMn/OVA and Alum/OVA groups increased antigen entry into PPs by a small amount. In contrast, OVA-positive areas in PPs of the MNC@CaMn/OVA(M) group were significantly increased. This further suggests that the simultaneous application of magnetic field and magnetic-driven nanorobots can promote the antigen to cross the mucosal layer and prolong the retention time. This would effectively promote antigen uptake and presentation. Subsequently, the distribution of antigen in the mesenteric lymph nodes was examined to detect the level of antigen being transported to secondary lymphoid organs. As shown in Fig. 7 c, the simultaneous application of magnetic field and magnetic-driven nanorobots promoted the entry of antigens into the mesenteric lymph nodes, which would help antigens to be presented to T cells, activate the T cells, and induce higher mucosal immune responses and systemic immune responses. Accordingly, we examined the activation and differentiation of T cells in two major secondary lymphoid organs (proximal mesenteric lymph nodes and distal spleen). Among them, the activation of cytotoxic CD8 + T lymphocytes is the important feature of cellular immunity, which has the function of directly killing pathogens. As shown in Figs. 7 d-f, the proportion of CD8 + T cells in the MNC@CaMn/OVA group did not increase significantly compared with that in the OVA group, whereas the proportion of CD8 + T cells in the MNC@CaMn/OVA(M) group significantly increase in the spleen and mesenteric lymph nodes. This may be due to the fact that the magnetic field can significantly increase the residence time of antigen-loaded magnetic-driven nanorobots at the inoculation site, recruiting more APCs and increasing the chances of antigen delivery, whereas magnetic-driven nanorobots release OVA and Mn 2+ in acidic lysosomal environments, which will further promote the cross-presentation of antigens, inducing a more pronounced CD8 + T cell proliferation and differentiation, and promoting the cellular immune response. In addition, the proliferation of splenocytes and lymphocytes also is important indicators of enhanced immune response after vaccination. Here, splenocytes and lymphocytes of immunized mice were collected for counting and the results are shown in Figs. 7 g&h. Splenocytes and lymphocytes were significantly increased in the MNC@CaMn/OVA(M) group compared to the OVA control group. This finding reveals the unique ability of magnetic-driven nanorobots in the presence of a magnetic field-promoting the effective crossing of antigens across the mucosal barrier and significantly enhancing the body's immune response. 3.8 Magnetic-driven nanorobots activate memory immune response The immune memory response is a hallmark feature of adaptive immunity and plays a crucial role in protecting the body from secondary attacks by pathogens [ 30 ]. To further evaluate the immune memory effect induced by oral vaccines prepared with magnetic-driven nanorobots, we further analyzed the proportion of memory T cells in the spleens of immunized mice. As shown in Figs. 8 a-c, oral vaccination with nanorobot vaccines significantly increased the number of CD4 + effector memory T cells (CD44 high CD62L low ) but not CD8 + effector memory T cells in the spleens of mice compared to the antigen-only group, which was attributed to the fact that nanorobot loading of the antigens could increase the retention of the vaccines in vivo to some extent. However, when nanorobot-vaccinated mice were simultaneously applied with a magnetic field, the number of both CD8 + and CD4 + effector memory T cells (CD44 high CD62L low ) was significantly increased in the spleens of the mice. This increase is attributed to the nanorobots' rapid crossing of the mucus layer under the action of the magnetic field, which increase the retention time of antigens in the intestine and provides crucial assistance in initiating immune responses and generating long-term anti-tumor immunity. The long-term benefits of immunotherapy require the generation of an antitumor memory effect, meaning that when immune mice are re-exposed to the same antigen, they can respond rapidly and effectively. Therefore, to assess whether treated mice can mount a cytotoxic response against tumor cells upon re-challenge with tumor antigens, we collected splenocytes from the immune mice and restimulated them with the antigen OVA for 48 hours. The supernatants were then collected and analyzed using enzyme-linked immunosorbent assay (ELISA) to measure the levels of cytokines (IFN-γ, TNF-α, IL-6, and IL-4). IFN-γ promotes the production of the antibody IgG2a and the differentiation of T cells into CTLs, making it an important cytokine in cellular immune responses[ 31 – 33 ]. Similarly, TNF-α is highly associated with antitumor immunity [ 34 – 36 ] and can cause hemorrhagic necrosis in solid tumors[ 37 ]. IL-6 also plays a role in the regulation of cellular immunity. As shown in Figs. 8 e-g, compared to OVA alone, the nanorobot vaccine group without magnetic field treatment only slightly increased the expression of antitumor-related cytokines (IFN-γ, TNF-α, and IL-6), while the nanorobot vaccine group with magnetic field treatment showed a significant increase in the expression of these antitumor-related cytokines, at 26.6 times, 2.5 times, and 1.9 times higher than the OVA group, respectively. This indicates that the combination of the nanorobot vaccine and magnetic field treatment maximizes the immune memory effect in mice, enabling a rapid response to re-stimulation with tumor antigens and release of inflammatory factors, thereby enhancing the antitumor immune response. Additionally, the combination of the nanorobot vaccine and magnetic field treatment also increased the expression of IL-4 in restimulated splenocytes (Fig. 8 h), activating a Th2 (type 2 helper T cell) immune response, which aids in promoting B cell differentiation and antibody production, particularly IgG1[ 38 , 39 ]. This is consistent with the previously mentioned IgG1 detection results (Fig. 5 d). 3.9 Tissue Safety of magnetic-driven nanorobot vaccines The biological safety of vaccine formulations is crucial for in vivo administration. On the fourth day after the last immunization, the major organs (ileum, stomach, heart, liver, spleen, lungs, and kidneys) were collected from the immunized mice to assess the tissue pathological toxicity of the vaccine formulation. As shown in Fig. 8 d, the simultaneous application of magnetic-driven nanorobots and magnetic fields does not lead to inflammation at the inoculation site (the small intestine) and does not cause damage to the gastric tissue. Furthermore, the simultaneous use of magnetic-driven nanorobots and magnetic fields does not cause significant pathological changes in major organs (heart, liver, spleen, lungs, and kidneys), indicating that magnet-driven nanorobots possess good biocompatibility and safety. 3.10 Antitumor immunotherapy using magnetic-driven nanorobot vaccines Oral administration of magnetic-driven nanorobot vaccines can effectively enhance mucosal and cellular immune responses. Therefore, it was necessary to evaluate the in vivo antitumor efficacy of oral vaccination. We subcutaneously injected B16-OVA tumor cells. When the tumor sizes reached approximately 50 mm³, the oral vaccine was administered every four days for a total of three doses (Fig. 9 a). The tumor growth curves showed that oral administration of OVA or MNC@CaMn/OVA did not significantly inhibit melanoma growth. However, the growth of melanomas in the magnetic-driven nanorobot vaccine group with magnetic field was markedly suppressed. Compared to the control group, the tumor volume in the magnet-driven nanorobot vaccine group with magnetic field was reduced by approximately 69% (Figs. 9 b&c). Photos of tumor-bearing mice are shown in Fig. 9 g, and the photos of the collected tumors are shown in Figs. 9 d. Compared to the tumors in control mice, the tumor sizes were in the magnet-driven nanorobot vaccine group with magnetic field was obviously reduced. Then, tumors collected from the mice were weighed, and the results are shown in Figs. 9 e. Compared to the tumors in normal mice (average weight: 1.48 g), the tumor weights in the magnetic-driven nanorobot vaccine group with magnetic field were significantly reduced, averaging just 0.58 g. In addition, the magnetic-driven nanorobot vaccine and magnetic field had no significant impact on the body weight of the mice (Fig. 9 f). These findings indicate that oral administration of the magnet-driven nanorobot vaccine under magnetic field effectively enhances antitumor immunotherapy. Certain biochemical indicators in the blood of melanoma-bearing mice (AST, ALT, and LDH) can reflect the efficacy and biosafety of the vaccine formulation[ 40 , 41 ]. Normal serum values of ALT, AST and LDH in mice ranged from 10.1–96.5 U/L, 36.3-235.5 U/L and 157.4-899.7 U/L, respectively. As shown in Figs. 9 h-j, the levels of ALT, AST and LDH in normal mice were within the normal range, whereas untreated melanoma-bearing mice had average ALT levels of 105.9 U/L, average AST levels of 1291.7 U/L, and average LDH levels of 5808.7 U/L, all far exceeding the normal range for mice. The average ALT levels in the magnet-driven nanorobot vaccine group with magnetic field were significantly reduced and within the normal range for mice. Moreover, the average AST and LDH levels in the magnet-driven nanorobot vaccine group with magnetic field were significantly lower compared to those in the untreated groups. This suggests that the magnet-driven nanorobot vaccine can reduce tumor size and maintain the normal function of mouse organs. Next, we further investigated the antitumor immune response activated by magnetic-driven nanorobot vaccine with magnetic field. The activation of CD8 + T cell releases interferons, perforin, and granzymes [ 42 ]. Therefore, assessing the infiltration of cytotoxic CD8 + T cells in tumor tissue is a critical indicator for evaluating the antitumor immune response. As shown in Fig. 10 a, CD8 + T cell staining of tumor tissue sections showed that the CD8 + T cell infiltration induced by the antigen alone and nanorobot vaccine without magnetic field groups was low in tumor tissues, whereas the magnetic-driven nanorobot vaccine with magnetic field obviously increased the level of CD8 + T cell infiltration in tumor tissues. This may be due to the magnetic field driving the nanorobots to rapidly cross the mucus layer, facilitating the interaction between the antigen and APCs in the lamina propria, enhancing antigen presentation, and aiding in the activation of CD8 + T cells, which further inhibits tumor growth. Another key phenomenon indicating enhanced tumor immune cycle reactions is the significant activation of CD8 + T cells in immune organs. Thus, detecting the proportion of CD8 + T cells in the spleen is very important. As shown in Figs. 10 b&c, the magnetic-driven nanorobot vaccine significantly increased the proportion of CD8 + T cells in the spleen compared to untreated tumor-bearing mice. This is important for enhancing systemic anti-tumor immune responses. Then, another mouse immune tissues - inguinal lymph nodes were collected and shown in Fig. 10 d, the sizes of bilateral inguinal lymph nodes obviously increased in the magnetic-driven nanorobot with magnetic field group compared to the control group, which indicates lymphocyte proliferation. All these results reconfirmed that the magnetic-driven nanorobot vaccine was effective in enhancing the anti-tumor immune response. Conclusion In order to promote the rapid and active crossing of the intestinal mucosal barrier by the vaccine and to prolong its residence time in the intestinal mucosa, we synthesized biodegradable magnetic-driven nanomotors (MNC@CaMn) loaded with antigens and constructed a magnetic-driven nanomotor vaccine delivery platform. Under the precise regulation of the magnetic field, the residence time of the vaccine in the intestines was significantly prolonged and exhibited the characteristics of swarming motility, which could rapidly aggregate and cross the intestinal mucus layer in a targeted manner, greatly facilitating antigen delivery and effectively inducing mucosal and systemic immune responses. The magnetic-driven nanomotor vaccine delivery system constructed in this study provides a new strategy for the development of efficient oral and mucosal vaccines. Declarations Conflict of Interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Foundation This work was granted by the National Natural Science Foundation of China (82404556), the National Natural Science Foundation of China (32471525), the Fundamental and Applied Basic Research Project of Guangzhou (SL2023A04J00799), and the General Project of Natural Science Foundation of Guangdong Province (2022A1515010715) Author Contribution L.H.: Methodology, Data curation, Software, Funding acquisition, Writing-original draft. X.S.: Investigation, Administration, Formal analysis, Data curation. Q.Z.: Formal analysis, Investigation. J.L.: Formal analysis, Investigation. W.C.: Formal analysis, Investigation. R.D.:Conceptualization, Supervision, Resources, Funding acquisition. Z.L.: Writing - review & editing, Supervision, Resources, Funding acquisition. Z.G.: Writing - review & editing, Project administration, Supervision, Resources. All authors reviewed the manuscript. References Vighi G, Marcucci F, Sensi L, Di Cara G, Frati F. Allergy and the gastrointestinal system. 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Carnosic acid impedes cell growth and enhances anticancer effects of carmustine and lomustine in melanoma. Biosci Rep 38(4) (2018). Stenger S, Hanson DA, Teitelbaum R, Dewan P, Niazi KR, Froelich CJ, Ganz T, Thoma-Uszynski S, Melián A, Bogdan C, Porcelli SA, Bloom BR, Krensky AM, Modlin RL. An antimicrobial activity of cytolytic T cells mediated by granulysin. Science. 1998;282(5386):121–5. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Video1.mp4 Video2.mp4 Video3.mp4 Video4.mp4 floatimage1.png Schema 1. Schematic representation of magnetic-driven nanorobots facilitating oral vaccine delivery across the intestinal mucosa to enhance mucosal and systemic immune responses. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5388438","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":374915586,"identity":"8c0d9142-8804-44cb-ad6c-c902a45ae9bc","order_by":0,"name":"Linghong Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsklEQVRIiWNgGAWjYFACHsbDIIqfmfnwA+I0ACFYi2Q7W5oBaVoMzvMoSBClxZ6998Dhgoo7dpsP8zAYMNTYRBO2hedcwuEZZ54lbzvMe+ABw7G03AaCWiRyDA7zth1ONjvMl2DA2HCYBC3GzTwGEiRpsTNgJlrLmTMGh3nOHE6QOAwM5ARi/MLe3mP4mKfisD1//+HDDz7U2BDWAgOJYJUJxCoHAXtSFI+CUTAKRsEIAwDePT5pT/FQ/QAAAABJRU5ErkJggg==","orcid":"","institution":"Jinan University","correspondingAuthor":true,"prefix":"","firstName":"Linghong","middleName":"","lastName":"Huang","suffix":""},{"id":374915587,"identity":"fdae2a21-8583-4323-b6e0-5ae99d0048c4","order_by":1,"name":"Xinyuan Sun","email":"","orcid":"","institution":"Guangzhou Institute of Urology, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinyuan","middleName":"","lastName":"Sun","suffix":""},{"id":374915588,"identity":"c86a8801-c2d0-48fb-bcb0-1461107a4f10","order_by":2,"name":"Quan Zhang","email":"","orcid":"","institution":"Guangzhou Institute of Urology, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Quan","middleName":"","lastName":"Zhang","suffix":""},{"id":374915589,"identity":"30269684-e620-49e7-a678-8dc3b2044837","order_by":3,"name":"Jun Long","email":"","orcid":"","institution":"Guangzhou Institute of Urology, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Long","suffix":""},{"id":374915590,"identity":"83414f0c-7b06-47b2-bbb5-085fcb24e4dc","order_by":4,"name":"Xuewu Chen","email":"","orcid":"","institution":"Guangzhou Institute of Urology, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xuewu","middleName":"","lastName":"Chen","suffix":""},{"id":374915591,"identity":"ce168f8d-a4ec-466e-94b0-f01f8bf36b3d","order_by":5,"name":"Renfeng Dong","email":"","orcid":"","institution":"South China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Renfeng","middleName":"","lastName":"Dong","suffix":""},{"id":374915592,"identity":"88373fac-5606-4dbf-b0b9-d6fe692b86ca","order_by":6,"name":"Zonghua Liu","email":"","orcid":"","institution":"Jinan University","correspondingAuthor":false,"prefix":"","firstName":"Zonghua","middleName":"","lastName":"Liu","suffix":""},{"id":374915593,"identity":"35e30882-ef75-4ed3-9b1c-d8151f1be460","order_by":7,"name":"Zhong Guo","email":"","orcid":"","institution":"Beijing Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zhong","middleName":"","lastName":"Guo","suffix":""}],"badges":[],"createdAt":"2024-11-04 13:23:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5388438/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5388438/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69328305,"identity":"bec5c935-f9ee-4b55-8028-88f908098c84","added_by":"auto","created_at":"2024-11-19 08:35:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":394089,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of MNC@CaMn nanorobots. The SEM images of (a) MNC and (b,c) MNC@CaMn nanorobots. (d) The EDS-mapping images of MNC@CaMn nanorobots. (e) The XRD spectrum of MNC@CaMn nanorobots. (f) The FT-IR spectrum of MNC@CaMn, OVA, and MNC@CaMn/OVA nanorobots. (g) The loading quantity of OVA by MNC@CaMn nanorobots. (h) The XPS full spectrum of the MNC@CaMn/OVA nanorobots. (i) XPS spectrum of N 1s in MNC@CaMn/OVA nanorobots. (j) The hysteresis line of MNC and MNC@CaMn nanorobots. (k) The magnetic properties of MNC@CaMn nanorobots. (l) The images of motors crossing artificial mucus under magnetic field.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5388438/v1/8df89abb59bcb92329b3d7fb.png"},{"id":69329244,"identity":"a098f978-da4c-4508-8db2-a5f030e9ddbd","added_by":"auto","created_at":"2024-11-19 08:43:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":600655,"visible":true,"origin":"","legend":"\u003cp\u003eThe swarm and navigational movement of MNC@CaMn nanorobots traveling through a maze containing (a) water, (b) DMEM, and (c) artificial mucus. (d) The enlarged version of the swarm and navigational movement of the nanorobots.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5388438/v1/d0a1e16bc5c214c7e6e17e1a.png"},{"id":69328303,"identity":"3ba889ea-87ca-4722-bd15-103d91f5b2fa","added_by":"auto","created_at":"2024-11-19 08:35:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":233798,"visible":true,"origin":"","legend":"\u003cp\u003eThe uptake of antigen by DCs. CLSM images of DC2.4 and BMDCs after incubation with nanorobot vaccines for 6 h. White scar bar: 20 μm, Black scar bar: 10 μm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5388438/v1/f5c74b7b2e98e1be1a27f5b9.png"},{"id":69328302,"identity":"197e003a-91b2-4f9f-9287-ccf65d1549df","added_by":"auto","created_at":"2024-11-19 08:35:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":330070,"visible":true,"origin":"","legend":"\u003cp\u003eStimulatory effects of BMDCs and BMDMs after co-incubation with MNC/CaMn nanorobot vaccines for 24 h. (a) The representative flow cytometry scatter plots of CD86, CD80, and MHC I molecules on the surface of BMDCs. (b) The representative flow cytometry scatter plots of CD206 and CD86 molecules on the surface of BMDMs. (c-d) Corresponding statistical graphs of CD86, CD80, and MHC I molecules on the surface of BMDCs. (e) Corresponding statistical graphs of CD206 and CD86 molecules on the surface of BMDMs.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5388438/v1/3a526642692bfdcf86ff29aa.png"},{"id":69329246,"identity":"b3378a62-4a4e-4004-ab9c-92d7c936b005","added_by":"auto","created_at":"2024-11-19 08:43:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":244802,"visible":true,"origin":"","legend":"\u003cp\u003eObservation of antigen at gastrointestinal sites and mesenteric lymph nodes. (a) Fluorescence intensity of antigens at gastrointestinal sites in mice, (b) and the corresponding average radiant efficiency from each group of mice. (c) Antigens in the gastrointestinal tract at 6 h and 24 h, and (d-e) the corresponding average radiation efficiencies in the stomachs, duodenums, jejunums, and lleums. (f) Observation of antigen at mesenteric lymph nodes from each group of mice, and (g) the corresponding average radiant efficiency.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5388438/v1/a03ad7eb6e5041b7e436c449.png"},{"id":69329524,"identity":"2c28fa56-079a-42a8-90fe-3e580d6147e7","added_by":"auto","created_at":"2024-11-19 08:51:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":581032,"visible":true,"origin":"","legend":"\u003cp\u003eThe immunization and immune response of magnetic-driven nanorobot vaccines. (a) Schematic diagram of the oral vaccine immunization. OVA-specific antibody titres for (b) IgG, (c) IgG1 and (d) IgG2a in serum. (e) Immunohistochemical images of antigens in the ileum after oral vaccination, brown areas are antigens. (f) Immunofluorescent images of DCs and macrophages in the ileum. (g-k) The OVA-specific antibody titres for IgA in fresh fecal supernatants at different times.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5388438/v1/022d26301cabe4181399108f.png"},{"id":69329249,"identity":"cd82203d-359f-484f-a730-5a7cc4654ecc","added_by":"auto","created_at":"2024-11-19 08:43:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":609432,"visible":true,"origin":"","legend":"\u003cp\u003eThe immune response of magnetic-driven nanorobot vaccines. (a) Immunofluorescence images of DCs and macrophages in the paired lymph nodes after oral vaccination. (b) Immunofluorescence images of DCs in the mesenteric lymph nodes. (c) Immunohistochemistry images of antigens in PPs and mesenteric lymph nodes. Proportion of CD8\u003csup\u003e+\u003c/sup\u003e/CD4\u003csup\u003e+\u003c/sup\u003e T cells in the spleen and mesenteric lymph nodes. (d) Representative flow scatter plots. (e-f) Statistical plots of the proportion of CD8\u003csup\u003e+\u003c/sup\u003e/CD4\u003csup\u003e+ \u003c/sup\u003eT cells in the spleen and mesenteric lymph nodes. Statistics of the cell counts in the (g) splenocytes and (h) lymphocytes.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5388438/v1/6ed4a72f36d95824ae99e04c.png"},{"id":69328317,"identity":"1b809730-ad10-4ba2-906c-2d1ce6eda646","added_by":"auto","created_at":"2024-11-19 08:35:35","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1223085,"visible":true,"origin":"","legend":"\u003cp\u003eProportion of CD8\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e immune memory T cells in the spleen. (a) Flow scatter plot. Statistical graph of the proportion of (b) CD8\u003csup\u003e+\u003c/sup\u003e and (c) CD4\u003csup\u003e+\u003c/sup\u003e immune memory T cells. (d) HE staining of mouse stomach, intestinal and major tissues, scar bar: 100 μm. Cytokine levels of (e) IFN-γ, (f) TNF-α, (g) IL-6 and (h) IL-4 in splenocyte supernatants.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-5388438/v1/fc9bb5875a66c5675b512294.png"},{"id":69328308,"identity":"6fbe2dcd-cdaa-406e-a10b-277259f825d5","added_by":"auto","created_at":"2024-11-19 08:35:34","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":504870,"visible":true,"origin":"","legend":"\u003cp\u003eAntitumor immune response activated by magnetic-driven nanorobots. (a) Schematic diagram of the anti-tumor treatment strategy of the oral vaccine. (b) Tumor growth curves of each mouse after oral vaccination. (c) Mean growth curves of the tumors of the mice. (d) Tumor photos of the mice. (e) Tumor weights of the mice. (f) Mean body weight curves of the mice. (g) The photos of melanoma-bearing mice. Serum concentrations of (h) AST, (i) ALT and (j) LDH in mice.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-5388438/v1/60c862dfd6ea20ce8445906a.png"},{"id":69329247,"identity":"64ca9508-ff16-42fa-abcf-ccce2fe5247d","added_by":"auto","created_at":"2024-11-19 08:43:34","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":611791,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Immunofluorescence images of the tumors of the mice with CD8\u003csup\u003e+\u003c/sup\u003e T. CD8\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e T cell ratios in the spleens of mice. (b) Flow scatter plot of (c) CD8\u003csup\u003e+\u003c/sup\u003e/CD4\u003csup\u003e+\u003c/sup\u003e T cell ratio statistic in the spleens. (d) Pictures of right and left draining lymph nodes in mice.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-5388438/v1/1d4d0db4b39c30b34895c6be.png"},{"id":70222666,"identity":"6e0a4745-662d-420e-b5bd-33030265dcbb","added_by":"auto","created_at":"2024-11-29 17:17:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6135567,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5388438/v1/6ab7ddd6-acd1-49a8-a5af-3c2ca2d4e088.pdf"},{"id":69328313,"identity":"e4f3bf45-3f5f-437d-88fd-89bddaa1f742","added_by":"auto","created_at":"2024-11-19 08:35:34","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":40760761,"visible":true,"origin":"","legend":"","description":"","filename":"Video1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5388438/v1/ba83149b74650ac5789f802a.mp4"},{"id":69328315,"identity":"968f8ad6-8d1a-49c8-825e-95a82bd329a1","added_by":"auto","created_at":"2024-11-19 08:35:34","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":41275400,"visible":true,"origin":"","legend":"","description":"","filename":"Video2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5388438/v1/c39207b45f53132e441008d1.mp4"},{"id":69329250,"identity":"23cb01ac-b355-4324-a05d-ea650e5184bf","added_by":"auto","created_at":"2024-11-19 08:43:34","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":41391254,"visible":true,"origin":"","legend":"","description":"","filename":"Video3.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5388438/v1/ea034a98186caa6c346f5b6a.mp4"},{"id":69328316,"identity":"6d731068-8550-484a-9acc-f6ea4361807b","added_by":"auto","created_at":"2024-11-19 08:35:35","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":41203379,"visible":true,"origin":"","legend":"","description":"","filename":"Video4.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5388438/v1/c2e93f933df927854395a13e.mp4"},{"id":69330723,"identity":"eb0dac13-497d-481e-9e5a-c66231662849","added_by":"auto","created_at":"2024-11-19 08:59:34","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":493233,"visible":true,"origin":"","legend":"\u003cp\u003eSchema 1. Schematic representation of magnetic-driven nanorobots facilitating oral vaccine delivery across the intestinal mucosa to enhance mucosal and systemic immune responses.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5388438/v1/a0e9df2a24de02535f2a62cf.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"The swarmable magnetic-driven nanorobots for facilitating trans- intestinal mucosal delivery of oral vaccines to enhance mucosal and systemic immune responses","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGiven that the gut-associated lymphoid tissue (GALT), the body's largest immune center, houses approximately 70% of all immune cells, oral vaccines have emerged as a cutting-edge strategy for triggering a strong immune response[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Compared with the traditional intramuscular or subcutaneous routes of vaccination, oral vaccines activate both mucosal and systemic immune responses[\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], offering advantages such as simplicity of administration, improved patient acceptance, and cost-effectiveness[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Polio and rotavirus vaccines, which are commercially available today, are successful examples of oral vaccines. However, the promotion of oral vaccines is constrained by the harsh environment of the gastrointestinal tract, specifically, two major challenges: first, the destruction of vaccine activity by gastric acid and digestive enzymes; and second, the continuous secretion of mucus from the intestinal mucosa forms a physical barrier, which, together with intestinal motility, restricts the effective absorption of vaccine components[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Advances in enteric capsule technology have effectively overcome the threat of gastric acid and digestive enzymes to vaccines, but the intestinal mucus layer remains a significant barrier to the efficiency of oral vaccine absorption.\u003c/p\u003e \u003cp\u003eInnovative delivery technologies are revolutionizing the way oral vaccines cross the mucus barrier. Targeting the hydrophobic and negatively charged properties of mucin in mucus, DNA vaccines have been delivered using cationic viscous polymers to improve their retention in the gut and enhance antitumor immune responses[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, the hydrophobic cationic polymers bind excessively to the mucus, which in turn impedes the normal diffusion of the antigen[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. To address this challenge, Zhang et al. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] utilized hydrophilic and slightly negatively charged \u0026ldquo;mucus-inert\u0026rdquo; polyethylene glycol (PEG) derivatives to encapsulate antigens and cell-penetrating peptides. In the mucus environment, the PEG coating dissociates in time, exposing a core rich in cell-penetrating peptides, which facilitates the smooth penetration of the antigen into the epithelial cells and enhances its bioavailability. Although these innovative strategies help to improve the ability of the vaccine to cross the mucus, they are all passive and difficult to overcome the strong resistance of the mucus layer. In the future, we will focus on the development of more efficient and active mucus-crossing technologies, aiming to enable oral vaccines to cross the mucus layer rapidly and precisely, and to enhance the efficiency of the immune response of oral vaccines.\u003c/p\u003e \u003cp\u003eMagnetic-driven technology has a growing potential in the biomedical field, enabling precise manipulation in a non-invasive manner, and effectively crossing biological tissue barriers[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Magnetic-driven nanorobots, with their tiny size, excellent autonomous mobility, and precise navigation performance, make them a preferred choice for drug delivery. For tissues such as the gastrointestinal tract, which has a large cavity, magnetic-driven nanorobots can easily realize all-around autonomous movement under the guidance of a local magnetic field, which opens up new possibilities for efficiently breaking through the mucus layer and accelerating drug delivery. Although the motion characteristics, functionalization, and manipulation of individual magnetic micro-nanorobots have been deeply explored, how to precisely manipulate the swarms of nanorobots to achieve on-demand drug and energy delivery is still a technical challenge to overcome[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Compared to single particles, the advantage of population control is that it significantly improves the dose and efficiency of drug delivery, while enhancing the image contrast for drug tracking and monitoring, providing a better solution for drug delivery visualization. Unfortunately, there is a scarcity of dedicated research on oral magnetic driven delivery systems. Zhang et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] used magnetically controlled microneedles guided by an external magnetic field to puncture the intestinal mucosa and successfully deliver insulin. However, direct puncture of the intestinal mucosa by microneedles may increase the risk of infection. Given this, the development of novel magnetic-driven nanorobots as vaccine carriers is expected to improve the efficiency of vaccines across the mucus barrier while avoiding the potential risk of infection.\u003c/p\u003e \u003cp\u003eTo overcome the mucus barrier and gastrointestinal peristalsis, and prolong the retention time of antigens in the intestine, we focused on the development of a technology capable of rapidly and actively crossing the intestinal mucus layer. In the present study, we prepared biodegradable magnetic-driven nanorobots (MNC@CaMn) loaded with antigen and constructed a magnetic-driven nanorobot vaccine. Under the precise regulation of an external magnetic field, this vaccine was able to exhibit a collective behavior similar to that of a swarm of bees, which rapidly assembled and oriented to traverse the intestinal mucus layer, significantly prolonging the retention time of antigens in the intestines, which in turn facilitated the effective delivery and presentation of antigens. In addition, the rough texture of the surface of the nanorobot ensured the stable loading of antigens, and the Mn\u003csup\u003e2+\u003c/sup\u003e contained in the particles acted as an adjuvant to activate the antigen-presenting cells in the spleen and mesenteric lymph nodes, thus realizing the dual activation of both efficient mucosal immunity and systemic immunity (Schema 1). This work provides a new perspective for the development of magnetic-driven nanorobots for oral and mucosal vaccine delivery systems.\u003c/p\u003e"},{"header":"2 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eCaCl\u003csub\u003e2\u003c/sub\u003e, Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e, FeCl\u003csub\u003e3\u003c/sub\u003e, MnCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO, and potassium citrate were purchased from Macklin (China). Mucin powder was purchased from Yuanye Bio-Technology Co., Ltd (China). Ovalbumin (OVA) was purchased from Sigma (USA). BCA kit, cell counting kit-8 (CCK8), Lyso-Tracker and DAPI were purchased from Beyotime Biotechnology (China), Ovalbumin antibody was purchased from LifeSpan BioSciences (US). Anti-IgG, anti-IgG1, anti-IgG2a and anti-IgA antibody were purchased Abcam (UK). The CD11C antibody, CD68 antibody and CD8-GB13429 were purchased from Servicebio (China). All flow cytometry antibody dyes and cytokine ELISA detection kits were purchased from BioLegend (USA). All female C57BL/6 mice (4\u0026ndash;6 weeks) used in the study were purchased from Beijing HFK Laboratory Animal Technology Co. (China). In addition, all animal experiments were eligible and approved by the Ethics Committee of Jinan University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation and characterization of MNC@CaMn nanorobots\u003c/h2\u003e \u003cp\u003ePreparation of magnetic nanoclusters (MNCs): First, potassium citrate (0.4 g) and FeCl\u003csub\u003e3\u003c/sub\u003e (0.65 g) were dissolved in ethylene glycol (40 mL) and vigorously stirred at room temperature for 1 h. Then, sodium acetate (1.2 g) was added and stirred for 30 min. The mixture was then transferred to a high-pressure reaction kettle and reacted at 200\u0026deg;C for 12 h. Finally, the product was collected, washed three times with ethanol and water, and stored by freeze-drying.\u003c/p\u003e \u003cp\u003ePreparation and characterization of MNC@CaMn nanorobots: First, CaCl\u003csub\u003e2\u003c/sub\u003e and MnCl\u003csub\u003e2\u003c/sub\u003e mixed solution (0.016 M, molar ratio of Ca\u003csup\u003e2+\u003c/sup\u003e/Mn\u003csup\u003e2+\u003c/sup\u003e = 1:1) was prepared using glycerol/water solution (1/1, v/v). Then, MNCs (5 mg) were added to the CaCl\u003csub\u003e2\u003c/sub\u003e and MnCl\u003csub\u003e2\u003c/sub\u003e mixed solution (10 mL) and stirred for 30 min. then NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e (10 mL, 0.16 M) in glycerol/water solution (1/1, v/v) was added to the mixture and stirred for 1 h at 50\u0026deg;C. and the precipitate was centrifuged and washed three times. Finally, the formed nanoparticles were characterized by scanning electron microscopy (SEM, Zeiss, Germany), transmission electron microscopy (TEM, JEM-2010HR, Japan), X-ray powder diffractometer (XRD, Rigaku, Japan), Fourier infrared spectroscopy (FT-IR, VERTEX70, Germany), laser nanoparticle sizer (Malvern, Britain) and vibrating sample magnetometer (VSM) (LakeShore, US). In addition, 150 mg of mucin was taken in 5 mL of deionized water to prepare artificial intestinal mucus. Then, 5 mg of magnetic nanorobots were added to this intestinal simulated mucosal fluid. The ability of the magnetic nanorobots to penetrate the mucus layer in the presence or absence of a magnetic field was observed and recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 The loading of OVA on MNC@CaMn nanorobots\u003c/h2\u003e \u003cp\u003eFirst, 1 mL of OVA solution (1 mg/mL) was prepared using saline. Then, the MNC@CaMn nanorobots (5 mg) were added to OVA solution (1 mL), and the suspension was stirred at room temperature. The free OVA in the solution was detected at different times using a BCA kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Swarm motion detection by MNC@CaMn nanorobots\u003c/h2\u003e \u003cp\u003eFirst, the labyrinth was modeled with transparent resin by using 3D printing technology, and then water, DMEM, or artificial mucus was added to the labyrinth. Subsequently, a suspension of MNC@CaMn nanorobots was dropped into the labyrinth. Next, a rotating permanent magnet was placed underneath the labyrinth to aggregate the MNC@CaMn nanorobots, and then the rotating magnet was moved to move the aggregated FMP through the labyrinth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Co-localization of magnetic-driven nanorobots in DCs\u003c/h2\u003e \u003cp\u003eFirst, the hind limb bones were isolated from female C57BL/6 mice. Then, the two distal bone ends were excised, and the bone marrow cells were flushed with RPMI 1640. The red blood cells in bone marrow cells were lysed and the remaining cells were centrifuged at 1500 rpm for 10 min. Subsequently, the cells were seeded into 6-well plates and cultured in RPMI 1640 medium (containing 20 ng/mL GM-CSF and 10 ng/mL IL-4) for 6 days. The medium was changed every two days. On day 6, immature BMDCs were obtained. Next, DC2.4 and BMDCs (5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/dish) were inoculated in cell culture dishes. After incubation, the cells were incubated with Cy5.5-OVA or MNC@CaMn/Cy5.5-OVA preparations for 6 h. Subsequently, the cells were incubated with Lyso-Green fluorescent dye for 2 h and fixed for 20 min, and then stained with DAPI dye solution for 5 min. Finally, the cells were observed with a confocal laser scanning microscope (CLSM, ZEISS, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Stimulation of BMDCs and BMDMs in vitro\u003c/h2\u003e \u003cp\u003eThe above immature BMDCs were inoculated into 24-well low-attachment surface plates (2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) and treated with PBS, OVA, MNC@CaMn/OVA, and LPS/OVA solution (OVA: 5 \u0026micro;g/well) for 24 h. Then, the fluorescent dye-labeled antibodies solutions (anti-CD11c-APC, anti-CD86-PerCP-Cy5.5, anti-CD80-PerCP-Cy5.5 and anti-H2Kb/SIINFEKL (MHC I)-PE) to detect DC maturity and the expression of OVA-specific MHC I with a flow cytometer (Beckman Coulter, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 In vivo evaluations of oral vaccine levels in the gastrointestinal tract and mesenteric lymph nodes\u003c/h2\u003e \u003cp\u003eFirst, MNC@CaMn nanorobots loaded with Cy5.5-OVA were used to prepare fluorescent magnetic-driven nanorobot vaccines. Subsequently, these nanorobot vaccines were administered by gavage to C57BL/6 female mice (4\u0026ndash;6 weeks old). Then, the mice were randomly divided into two groups (n\u0026thinsp;=\u0026thinsp;4), one group of mice was magnetically treated and the other group was not magnetically treated (100 \u0026micro;g Cy5.5-OVA/mouse, 1 mg MNC@CaMn/mouse). The fluorescence intensity in the gastrointestinal tract area of mice was detected with a small animal bioluminescence imaging system (IVIS Lumina III, PerkinElmer, USA) at 1, 6 and 24 h after gavage. In addition, at 6 and 24 h after gavage, the mouse mesenteric lymph nodes and gastrointestinal tract tissues were collected and their fluorescence intensity were examined with the small animal bioluminescence imaging system. All of the above mice were starved overnight before gavage, and 100 \u0026micro;L of 1% NaHCO\u003csub\u003e3\u003c/sub\u003e solution was administered orally 30 min before gavage to neutralize gastric acid.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 In vivo immunization assessment of oral vaccines\u003c/h2\u003e \u003cp\u003eC57BL/6 female mice (4\u0026ndash;6 weeks old) were randomly divided into 5 groups (n\u0026thinsp;=\u0026thinsp;5) and gavaged with 100 \u0026micro;L of OVA, MNC@CaMn/OVA, MNC@CaMn/OVA (M), and Alum/OVA solution (OVA: 100 \u0026micro;g/mouse). The mice were vaccinated four with interval of 4 days. Then, 0.05 g of fresh feces were collected from mice on days 0, 8, 12 and 16 and made into a suspension of 0.1 g/mL. Subsequently, the suspension was centrifuged at 12,000 rpm for 30 min to collect supernatant. On the 4th day after the last inoculation, serum was separated from the blood of the experimental mice and stored at -20℃. Subsequently, the splenocytes and lymphocytes were isolated from collected spleens and mesenteric lymph nodes of mice. All of the above mice were starved overnight before gavage. The mice were gavaged with 100 \u0026micro;L of 1% NaHCO\u003csub\u003e3\u003c/sub\u003e solution to neutralize the gastric acid 30 min prior to gavage of the nanorobot.\u003c/p\u003e \u003cp\u003eDetection of the ability of nanorobots to pass through the intestinal mucosa: the mouse intestines were removed, fixed with paraformaldehyde, paraffin-embedded, sectioned and immunohistochemically stained, and the mouse intestines were observed by a fluorescence microscope (DMRA2, Leica, Germany).\u003c/p\u003e \u003cp\u003eDC and macrophage recruitment and migration assays: the mouse ileal tissues and peyer patchs were collected, fixed with 4% paraformaldehyde, sectioned, and stained by immunofluorescence using CD11C and CD68 antibody dyes. Then, the recruitment of DCs and macrophages in the mouse ileum and their migration to the peyer patchs were observed with the fluorescence microscope.\u003c/p\u003e \u003cp\u003eImmunohistochemical analysis of peyer patchs and mesenteric lymph nodes: the mouse ileal tissues, peyer patchs, and mesenteric lymph nodes were collected and fixed with 4% paraformaldehyde for immunohistochemical analysis. The distribution of antigen protein OVA in the ileal tissues, peyer patchs, and mesenteric lymph nodes was observed with the fluorescence microscope.\u003c/p\u003e \u003cp\u003eDetection of serum and mucosal antibody titers: the enzyme-linked immunosorbent assay (ELISA) technique was used to detect the levels of OVA-specific antibodies (IgG, IgG1 and IgG2a) in the serum of experimental mice as well as the levels of IgA antibodies in the fecal supernatant.\u003c/p\u003e \u003cp\u003eDetection of cytokines secreted by splenocytes: the obtained splenocytes were inoculated into 12-well plates (5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) and then stimulated with antigen OVA solution (final concentration: 25 \u0026micro;g/mL) for 60 h. Subsequently, the cellular supernatants were collected to detect the secretion levels of cytokines IFN-γ, TNF-α, IL-6, and IL-4 by using an ELISA kit.\u003c/p\u003e \u003cp\u003eDetection of T-cell ratio in splenocytes and lymphocytes: the Splenocytes and lymphocytes (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mouse) were extracted from the above mice and stained with fluorescent dye-labeled antibodies (anti-CD3-APC, anti-CD8α-PerCP-Cy5.5 and anti-CD4-FITC). Subsequently, the cells were washed with PBS and resuspended. Finally, the cells were detected with the flow cytometer.\u003c/p\u003e \u003cp\u003eDetection of immune memory T-cell ratio: the Splenocytes and lymphocytes (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mouse) were extracted from the above mice and stained with fluorescent dye-labeled antibodies (anti-CD62L-APC, anti-CD44-PE, anti-CD8α-PerCP-Cy5.5 and anti-CD4-FITC). Subsequently, the cells were washed with PBS and resuspended. Finally, the cells were detected with the flow cytometer.\u003c/p\u003e \u003cp\u003eEvaluation of histocompatibility of vaccine formulations: the Hearts, livers, spleens, lungs, stomachs, ileums and kidneys of these experimental mice were collected and fixed with 4% paraformaldehyde. Subsequently, tissue sections were made from these organs and stained with hematoxylin-eosin (H\u0026amp;E) staining solution. Finally, the fluorescence microscope was used to observe the sections.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 In vivo antitumor immunological evaluation of oral vaccines\u003c/h2\u003e \u003cp\u003eC57BL/6 female mice (4\u0026ndash;6 weeks old) were randomized into 5 groups (n\u0026thinsp;=\u0026thinsp;5). On day 0, B16-OVA cells were injected subcutaneously into the left dorsum of each mouse. On days 7, 11, and 15, mice were orally inoculated with 100 \u0026micro;L of Saline, OVA, MNC@CaMn/OVA, or MNC@CaMn/OVA (M) formulations, and their body weights and tumor volumes were monitored every 1 day. On day 19 after tumor inoculation, the spleens of mice were collected and the lymph nodes and tumors were drained.\u003c/p\u003e \u003cp\u003eFirst, the collected tumors were sectioned and stained with CD8\u003csup\u003e+\u003c/sup\u003e T cell staining solution, and the infiltration of CD8\u003csup\u003e+\u003c/sup\u003e T cells in the mouse tumors was observed by the fluorescence microscope. Subsequently, splenocytes (1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/one) were collected from the spleen, stained with APC-anti-CD3, Percp-Cy5.5-anti-CD8a and FITC-anti-CD4, and detected by the flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism 5 and Origin software were used to statistically analyze the experimental results, and one-way ANOVA test was used to analyze the differences between the experimental groups. The experimental data were expressed as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Where \u003cem\u003e*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e, \u003cem\u003e**p\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e and \u003cem\u003e***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e were used to indicate significant differences.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Preparation and characterization of magnetic-driven nanorobots\u003c/h2\u003e \u003cp\u003eFirst, magnetic nanoclusters (MNCs) were prepared by hydrothermal method. Subsequently, calcium-doped manganese carbonate was further deposited on the MNC surface with Mn\u003csup\u003e2+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e and sodium bicarbonate to obtain MNC@CaMn nanorobots with core-shell structure. This synthesis strategy is a green chemistry reaction. The SEM images demonstrated that the MNCs had a spherical morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea), whereas the MNC@CaMn nanoparticles displayed an ellipsoidal morphology with sizes of approximately 200 nm and 600 nm, respectively. (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u0026amp;c). The elemental analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed) confirmed the presence of calcium (Ca), manganese (Mn), carbon (C) and oxygen (O) in the nanorobots. Subsequent XRD patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee) showed that the diffraction peaks of MNC@CaMn nanorobots at 2θ = 35.18°, 42.61°, 56.68°, and 62.40° corresponded to the (311), (400), (511), and (440) crystal planes of MNC nanoparticles in the standard XRD patterns[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], respectively; while the diffraction peaks at 2θ = 23.31°, 29.92°, 40.11°, and 49.29° correspond to the (012), (104), (113), and (116) crystal planes of MnCO\u003csub\u003e3\u003c/sub\u003e in the standard XRD patterns. All these results indicate the successful preparation of MNC@CaMn nanorobots.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, nanorobot-loaded model antigen OVA was used to evaluate its antigen loading efficiency. The infrared spectrum (IR) was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, the IR spectrum of MNC@CaMn showed stretching vibration absorption peaks of Fe-O-Fe near 581 cm\u003csup\u003e− 1\u003c/sup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]and bending vibration absorption peaks of CO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2−\u003c/sup\u003e near 869 cm\u003csup\u003e− 1\u003c/sup\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. While the IR spectrum of MNC@CaMn/OVA exhibit a new characteristic peak near 1650 cm\u003csup\u003e− 1\u003c/sup\u003e upon adsorption of protein antigen, primarily attributed to the stretching vibration of C = O in the amide group of the protein[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Broad peaks corresponding to N-H stretching vibrations were observed in the range of 3000–3750 cm\u003csup\u003e− 1\u003c/sup\u003e, while peaks associated with C-H stretching vibrations were detected in the range of 2700–3000 cm\u003csup\u003e− 1\u003c/sup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This indicates that the MNC@CaMn nanorobots can effectively adsorb antigen OVA. Additionally, the BCA assay was employed to quantify the antigen loading capacity of MNC@CaMn nanorobots at different time points, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg. The antigen loading capacity of MNC@CaMn nanorobots gradually increased over time, reaching maximum adsorption capacity (approximately 200 µg/mg) within 12 h and maintaining stable loading over an extended period. This will help in co-delivery of antigen and adjuvant. Furthermore, the XPS full spectrum of the MNC@CaMn/OVA nanorobots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh) revealed characteristic binding energy peaks for N 1s confirming the presence of N elements in the MNC@CaMn/OVA nanorobots. Elemental spectra (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei) further supported these findings, with peaks at 399.7 and 400.3 eV corresponding to the N 1s of OVA, indicating that MNC@CaMn nanorobots can effectively adsorb protein antigen.\u003c/p\u003e \u003cp\u003eIn addition, the vibrating sample magnetometer (VSM) and an NdFeB magnet block were used to examine the magnetic properties of MNC@CaMn nanorobots. As shown in the hysteresis line in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ej, both MNC and MNC@CaMn nanorobots exhibit superparamagnetism with coercivity close to zero. Their saturation magnetization strengths were 18.40 and 65.56 emu/g, respectively. Subsequently, the dispersed MNC@CaMn nanorobots rapidly aggregated when the NdFeB magnet was attached to the vial wall and completely aggregated within 10 s (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek). These results indicate that the MNC@CaMn nanorobots maintain good magnetic properties. Given that this project plans to use magnetic-driven nanorobots loaded with antigens for trans-intestinal mucus transport, we prepared artificial mucus using mucin to evaluate the trans-mucus ability of magnetic-driven nanorobots in vitro. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003el, the artificial mucus was relatively viscous and the nanorobots could not effectively traverse the artificial mucus without magnet guidance. However, with magnet guidance, the nanorobots could rapidly traverse the artificial mucus, suggesting that the magnetic-driven nanorobots can effectively cross the mucus layer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Navigational locomotion of nanorobots\u003c/h2\u003e \u003cp\u003eMagnetic driven and control technology has attracted much attention due to its remarkable potential for biomedical applications[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The advantage of this technology lies in the ability of the magnetic field to penetrate deep into biological tissues with virtually no side effects. For performing specific tasks in the complex intestinal environment, nanorobots must be highly intelligent in order to work effectively in the intestine. Compared to a single magnetic-driven nanorobot, swarm motions of nanorobots show greater advantages in achieving efficient drug delivery. In this study, we designed a complex labyrinth experiment to simulate the navigational ability of MNC@CaMn nanorobots in the intestine. The labyrinth is made of transparent resin material and fabricated by 3D printing technology. In the experiment, we used a rotating magnetic field to drive the nanorobots to demonstrate its clustering effect. Driven by the rotating magnetic field, the nanorobots rapidly aggregated to form a microcolony like a swarm of bees, and successfully completed a variety of continuous motions including straight-line advancement and turning (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and Supporting Information Video S1). In addition, the nanorobots also demonstrated its excellent navigation performance in a channel containing DMEM cell culture medium and artificial mucus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u0026amp;c and Supporting Information Videos S2\u0026amp;S3). These experimental results show that the nanorobots can efficiently traverse the mucus layer and realize the intended movement path even in a tiny and structurally complex environment. Further observations revealed that in the zoomed-in video (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed and Supporting Information Video S4), multiple hydrodynamic vortices are generated around the nanorobots when a rotating magnetic field is applied. There are interactions between these vortices that cause the nanorobots to be attracted to each other, thus causing the entire cluster to narrow down and all the nanorobots to be dynamically confined to a smaller area like a whole. This phenomenon not only facilitates the control of the cluster of nanorobots, but also simplifies the process of transporting them to the target location. Effective manipulation and precise positioning of the nanorobots can be achieved through simple magnetic field adjustments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Nanorobots facilitate antigen delivery to DCs\u003c/h2\u003e \u003cp\u003eAntigen uptake by antigen-presenting cells (APCs) is a critical first step in initiating antigen-specific immune responses[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. DCs, as the most specialized APCs, capture exogenous antigens, up-regulate co-stimulatory molecules to promote DCs maturation and migrate to lymph and spleen to deliver antigens to T cells via MHC molecules and activate T cells[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. To investigate the distribution of MNC@CaMn nanorobots loaded with antigen in APCs and their effects on cellular functions, we employed confocal laser scanning microscopy (CLSM) to observe the distribution of nanorobots loaded with Cy5.5-labeled OVA (Cy5.5-OVA) in DC2.4 and BMDCs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-d, Cy5.5-OVA alone entered the lysosomes of DC2.4 and BMDCs in relatively small amounts, while the nanorobots loaded with Cy5.5-OVA significantly increased the overlap of red fluorescence of antigens with the green fluorescence of lysosomes in DC2.4 and BMDCs. This result suggests that the nanorobots significantly increases the uptake efficiency of antigen by DCs. In addition, we noted that in the antigen alone group, BMDCs presented a typical rounded appearance, indicating that they were immature. Whereas, after stimulation with the nanorobots, BMDCs transformed into a polypod-like morphology (red arrows in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), which suggests that BMDCs were stimulated to mature. Together, these results confirm that the antigen loaded by the nanorobots significantly enhances the uptake of antigen by DCs and promotes the maturation of DCs, thus providing favorable conditions for the subsequent immune response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Nanorobot vaccines promote BMDC maturation and antigen presentation and stimulate BMDM polarization towards M1-type macrophages\u003c/h2\u003e \u003cp\u003eAPCs are the cells in the organism that have the ability to uptake, process and present antigen information and induce immune responses in T and B cells. They mainly include dendritic cells and macrophages. The high expression of exogenous antigenic peptide-specific MHC class I molecules in DCs implies that antigens are cross-presented, which is essential for activating anti-tumor immunity[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In addition, macrophages are similarly activated by antigen and differentiate into M1-type and M2-type macrophages. M1-type macrophages have strong tumor-killing and antigen-presenting abilities, which can enhance anti-tumor effects by activating anti-tumor T-cell responses. Therefore, reversal of immunosuppressive M2-type macrophages to M1-type macrophages is an important strategy to enhance anti-tumor immunotherapy[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In this study, isolated BMDCs were treated with MNC@CaMn nanorobot vaccines for 24 h and analyzed for the expression of OVA-specific MHC I, MHC II, CD80 and CD86 molecules. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec\u0026amp;d, OVA alone only slightly increased CD80/CD86 co-expression and hardly increased MHC I expression, indicating that OVA alone did not significantly promote BMDC maturation and MHC I-mediated antigen presentation. In contrast, antigen loading by the nanorobots significantly increased CD80/CD86 co-expression and MHC I expression, suggesting that the nanorobot vaccines significantly BMDC maturation and MHC I-mediated antigen presentation. This was attributed to the role of Mn\u003csup\u003e2+\u003c/sup\u003e in the nanorobots that could significantly increase cellular immune responses. Subsequently, after macrophages BMDMs were extracted from mouse bone marrow and induced to differentiate into M2-type macrophages using IL4, the proportion of M2-type macrophages that were reversed to M1-type macrophages was analyzed by treating with the nanorobot vaccines for 24 h. The results were shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb\u0026amp;e, CD206 molecules were significantly down-regulated while CD86 molecules were significantly up-regulated in the nanorobot vaccines group compared to the NC and OVA groups. Its CD86/CD206 ratio was close to that of the positive control LPS, suggesting that the nanorobots could significantly promote the reversal of M2-type macrophages to M1-type macrophages, which would significantly enhance the anti-tumor immune response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5 Magnetic fields promote retention of nanorobot vaccines in intestinal tissues and migration to mesenteric lymph nodes\u003c/b\u003e \u003c/p\u003e \u003cp\u003eLonger retention of antigen at the inoculation site and migration to peripheral immune organs can promote sustained immune stimulation and thus enhance the immune response. Here, the role of magnetic-driven nanorobots on antigen retention and migration in gastrointestinal tissues was investigated. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u0026amp;b, at 6 h after immunization, the fluorescence in the abdomen of mice with nanorobot without magnetic field treatment decreased rapidly, and only weak fluorescence could be observed at 24 h. However, the fluorescence in the abdomen of mice showed a stronger fluorescence signal when the nanorobot vaccines was treated with a magnetic field for 6 h. This is due to the fact that the originally dispersed nanorobots in the gastrointestinal tissues were aggregated by the magnetic field and concentrated to the abdominal surface of the mouse gastrointestinal tract, resulting in the detection of a stronger fluorescence signal. Moreover, when the magnetic field was withdrawn, the mouse inoculation site still contained a large amount of fluorescent signals at the 24 h. This suggests that the antigen-loaded MNC/CaMn nanorobots can achieve longer antigen retention time in the gastrointestinal tissues under magnetic field. At 6 h and 24 h after immunization, the gastrointestinal tissues and mesenteric lymph nodes of mice were isolated to further detect the retention and migration of OVA. The gastrointestinal tissues are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-e, and the OVA content in the gastrointestinal tract was significantly higher at 24 h in the magnetic field-treated group, compared with the no magnetic field group. The OVA content in the gastrointestinal tract was significantly higher in the magnetic field-treated group. This future indicates that the magnetic field prolonged the residence time of antigen in the gastrointestinal tissues. Subsequently, the results of mesenteric lymph nodes were shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef\u0026amp;g. At both 6 h and 24 h, the OVA content in the mesenteric lymph nodes of the group with magnetic field was higher than that of the group without magnetic field. This suggests that the magnetic field increases the retention time of the nanorobot vaccine at the vaccination site and also helps to promote antigen migration to the mesenteric lymph nodes, which contributes to the immune response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.6 Magnetic-driven nanorobots cross the mucus layer and recruit APCs to increase mucosal immune response\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eIn this study, magnetic-driven nanorobots were orally administered to the mice, and a vortex magnetic field was applied to their abdomens to test whether the nanorobots could successfully traverse the intestinal fluid and stimulate both mucosal and systemic immune responses. The vaccine administration schematic is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea. Antibody titer is a key indicator of the level of immune response. Mucosal vaccination induces not only systemic IgG antibodies, but also secretion of IgA antibodies from the mucosal surface[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, the levels of OVA-specific IgG, IgG1 and IgG2a antibodies in the serum of immunized mice and IgA antibodies in fresh feces were detected by ELISA. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-d, the Alum/OVA group did not significantly increase the levels of systemic IgG, IgG1 and IgG2a antibodies compared to the OVA alone group, suggesting that it is difficult to augment the immune response to an oral vaccine using only a simple aluminum adjuvant. In addition, the loading of OVA by the MNC/CaMn nanorobots only significantly increased the serum levels of IgG1 antibodies and did not significantly enhance the serum levels of IgG and IgG2a antibodies compared to the OVA alone group. However, nanorobots with magnetic fields significantly increased the expression of antigen-specific antibodies IgG and IgG2a compared to nanorobots without magnetic fields. This suggests that that magnetic nanorobots significantly increase the immune response, especially the cellular immune response, in the presence of a magnetic field. Moreover, the results of IgA antibody detection in fresh feces are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg-k. After immunization with OVA alone, the level of IgA antibody increases rapidly, then decreases sharply after the 8th day, and it cannot be maintained for a long period of time to induce the production of a high level of IgA antibody, and the MNC/CaMn/OVA nanorobot vaccines without magnetic field also didn’t effectively increase the level of IgA antibody. In contrast, the group of MNC/CaMn/OVA nanorobot vaccines with magnetic field showed a rapid increase in IgA antibody levels within 8 d and did not decrease significantly on both the 12th and 16th d. This suggests that oral administration of the MNC/CaMn/OVA nanorobot vaccines alone is not sufficient to increase the level of mucosal immune response to the antigens, which may be attributed to the fact that, in the absence of a magnetic field, the magnetic-driven nanorobot vaccines cannot traverse the mucosal layer of the gastrointestinal tract and cannot be uptaken by the immune cells in the epithelium of the gastrointestinal tract, whereas application of a magnetic field induces the magnetic-driven nanorobot vaccines to traverse the mucosal layer actively and rapidly, and prolongs their residence times in the gastrointestinal tract, which will be conducive to the production of mucosal and systemic antibodies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe small intestine is the target site for all currently approved oral vaccines and most studies reported[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Unlike the lining of the oral and gastric cavities, the intestinal mucosa recognizes foreign antigens and transports them through the mucosal barrier to the immune system[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Therefore, the oral vaccine in this research mainly chooses the small intestine as the object of analysis. Nanorobot vaccines passing through a magnetic field can increase the retention of antigens at the inoculation site. However, whether the nanorobot successfully carries antigens across the intestinal mucus layer in the presence of a magnetic field still requires further testing. Next, immunohistochemical analysis was performed to further detect the level of antigen crossing the intestinal mucus layer. The results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, OVA-positive areas (brown areas) were rarely observed in the OVA-alone group, whereas a large number of OVA-positive regions appeared in the intestinal mucus after the antigen-loaded MNC/CaMn nanorobots were treated with a magnetic field, and could be detected in the submucosal layer in the intestinal mucosa. This further suggests that the combined use of magnetic field and magnetic-driven nanorobots helps to prolong the residence time of the antigen in the intestine and promotes the penetration of the antigen into the intestinal mucosal layer, which would contribute to the uptake of the antigen by the APCs.\u003c/p\u003e \u003cp\u003eThe foundation of mucosal immunity is that APCs recruit and capture antigens at mucosal inductive sites (such as PPs), and then migrate through lymphatics to local mucosal-draining lymph nodes (such as mesenteric lymph nodes). Here, APCs present antigens to T cells, initiating both mucosal and systemic immune responses, while also generating antigen-specific memory cells and antibody-secreting cells, thereby establishing a long-lasting effective immune response against specific antigens[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, the successful recruitment of immune cells at the mucosal induction site is crucial for the mucosal immune response. We first observed the recruitment of APCs throughout the intestinal mucosal site, and the results, as shown by immunofluorescence staining images in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef, showed that the OVA alone and Alum/OVA groups recruited fewer DCs and macrophages, while the MNC/CaMn/OVA(M) group recruited more DCs and macrophages. It may be due to the fact that, the magnetic field effectively increased the retention and transmucosal layer efficiency of the magnetic-driven nanorobots loaded with antigen at the inoculation site, stimulated the intestinal epithelial tissue, and recruited more APCs. Next, APCs recruited in local mucosal-draining lymph nodes (such as PPs) were examined. The results were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, there were fewer DCs and macrophages in the PPs in the OVA alone and Alum/OVA groups, while there were significantly more DCs and macrophages in the PPs of the MNC@CaMn/OVA (M) group, which may be attributed to the fact that MNC@CaMn/OVA can rapidly aggregate and rapidly cross the intestinal mucus layer under magnetic field, stimulating the intestinal mucosa and recruiting more DCs and macrophages. This would help APCs to take up antigen and be stimulated to mature and accelerate migration into mesenteric lymph nodes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.7 The migration of APCs to mesenteric lymph nodes promotes antigen presentation\u003c/h2\u003e \u003cp\u003eAfter uptake of antigens, APCs need to migrate to lymph nodes for presenting antigens to T lymphocytes, which trigger an immune response. Although macrophages in APCs can also present antigens, their main functions are phagocytosis and antigen presentation in tissues, rather than migrating to immune organs to activate T cells[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Therefore, it is mainly activated DCs in APCs that have the function of migrating to secondary lymphoid organs and activating T cells[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, DCs in mesenteric lymph nodes, an important secondary lymphoid organ in mucosal immunity to oral vaccines, were examined and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb. There were significantly more DCs in the mesenteric lymph nodes in the MNC@CaMn/OVA (M) group compared to the OVA alone and Alum/OVA groups, suggesting that activated DCs migrated to the mesenteric lymph nodes in greater quantities, which is essential for the activation of mucosal immune responses by oral vaccines.\u003c/p\u003e \u003cp\u003eNext, immunohistochemical analyses were performed to further examine the level of antigen being transported to the mesenteric lymph nodes after uptake by APCs. First, the level of antigen enrichment in mucosal inductive sites PPs was examined. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, inoculation with OVA alone resulted in less OVA-positive areas (brown areas) in PPs, whereas MNC@CaMn/OVA and Alum/OVA groups increased antigen entry into PPs by a small amount. In contrast, OVA-positive areas in PPs of the MNC@CaMn/OVA(M) group were significantly increased. This further suggests that the simultaneous application of magnetic field and magnetic-driven nanorobots can promote the antigen to cross the mucosal layer and prolong the retention time. This would effectively promote antigen uptake and presentation.\u003c/p\u003e \u003cp\u003eSubsequently, the distribution of antigen in the mesenteric lymph nodes was examined to detect the level of antigen being transported to secondary lymphoid organs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, the simultaneous application of magnetic field and magnetic-driven nanorobots promoted the entry of antigens into the mesenteric lymph nodes, which would help antigens to be presented to T cells, activate the T cells, and induce higher mucosal immune responses and systemic immune responses. Accordingly, we examined the activation and differentiation of T cells in two major secondary lymphoid organs (proximal mesenteric lymph nodes and distal spleen). Among them, the activation of cytotoxic CD8\u003csup\u003e+\u003c/sup\u003e T lymphocytes is the important feature of cellular immunity, which has the function of directly killing pathogens. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed-f, the proportion of CD8\u003csup\u003e+\u003c/sup\u003e T cells in the MNC@CaMn/OVA group did not increase significantly compared with that in the OVA group, whereas the proportion of CD8\u003csup\u003e+\u003c/sup\u003e T cells in the MNC@CaMn/OVA(M) group significantly increase in the spleen and mesenteric lymph nodes. This may be due to the fact that the magnetic field can significantly increase the residence time of antigen-loaded magnetic-driven nanorobots at the inoculation site, recruiting more APCs and increasing the chances of antigen delivery, whereas magnetic-driven nanorobots release OVA and Mn\u003csup\u003e2+\u003c/sup\u003e in acidic lysosomal environments, which will further promote the cross-presentation of antigens, inducing a more pronounced CD8\u003csup\u003e+\u003c/sup\u003e T cell proliferation and differentiation, and promoting the cellular immune response.\u003c/p\u003e \u003cp\u003eIn addition, the proliferation of splenocytes and lymphocytes also is important indicators of enhanced immune response after vaccination. Here, splenocytes and lymphocytes of immunized mice were collected for counting and the results are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg\u0026amp;h. Splenocytes and lymphocytes were significantly increased in the MNC@CaMn/OVA(M) group compared to the OVA control group. This finding reveals the unique ability of magnetic-driven nanorobots in the presence of a magnetic field-promoting the effective crossing of antigens across the mucosal barrier and significantly enhancing the body's immune response.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Magnetic-driven nanorobots activate memory immune response\u003c/h2\u003e \u003cp\u003eThe immune memory response is a hallmark feature of adaptive immunity and plays a crucial role in protecting the body from secondary attacks by pathogens [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. To further evaluate the immune memory effect induced by oral vaccines prepared with magnetic-driven nanorobots, we further analyzed the proportion of memory T cells in the spleens of immunized mice. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea-c, oral vaccination with nanorobot vaccines significantly increased the number of CD4\u003csup\u003e+\u003c/sup\u003e effector memory T cells (CD44\u003csup\u003ehigh\u003c/sup\u003e CD62L\u003csup\u003elow\u003c/sup\u003e) but not CD8\u003csup\u003e+\u003c/sup\u003e effector memory T cells in the spleens of mice compared to the antigen-only group, which was attributed to the fact that nanorobot loading of the antigens could increase the retention of the vaccines in vivo to some extent. However, when nanorobot-vaccinated mice were simultaneously applied with a magnetic field, the number of both CD8\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e effector memory T cells (CD44\u003csup\u003ehigh\u003c/sup\u003e CD62L\u003csup\u003elow\u003c/sup\u003e) was significantly increased in the spleens of the mice. This increase is attributed to the nanorobots' rapid crossing of the mucus layer under the action of the magnetic field, which increase the retention time of antigens in the intestine and provides crucial assistance in initiating immune responses and generating long-term anti-tumor immunity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe long-term benefits of immunotherapy require the generation of an antitumor memory effect, meaning that when immune mice are re-exposed to the same antigen, they can respond rapidly and effectively. Therefore, to assess whether treated mice can mount a cytotoxic response against tumor cells upon re-challenge with tumor antigens, we collected splenocytes from the immune mice and restimulated them with the antigen OVA for 48 hours. The supernatants were then collected and analyzed using enzyme-linked immunosorbent assay (ELISA) to measure the levels of cytokines (IFN-γ, TNF-α, IL-6, and IL-4). IFN-γ promotes the production of the antibody IgG2a and the differentiation of T cells into CTLs, making it an important cytokine in cellular immune responses[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e–\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Similarly, TNF-α is highly associated with antitumor immunity [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e–\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and can cause hemorrhagic necrosis in solid tumors[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. IL-6 also plays a role in the regulation of cellular immunity. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee-g, compared to OVA alone, the nanorobot vaccine group without magnetic field treatment only slightly increased the expression of antitumor-related cytokines (IFN-γ, TNF-α, and IL-6), while the nanorobot vaccine group with magnetic field treatment showed a significant increase in the expression of these antitumor-related cytokines, at 26.6 times, 2.5 times, and 1.9 times higher than the OVA group, respectively. This indicates that the combination of the nanorobot vaccine and magnetic field treatment maximizes the immune memory effect in mice, enabling a rapid response to re-stimulation with tumor antigens and release of inflammatory factors, thereby enhancing the antitumor immune response. Additionally, the combination of the nanorobot vaccine and magnetic field treatment also increased the expression of IL-4 in restimulated splenocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eh), activating a Th2 (type 2 helper T cell) immune response, which aids in promoting B cell differentiation and antibody production, particularly IgG1[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This is consistent with the previously mentioned IgG1 detection results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Tissue Safety of magnetic-driven nanorobot vaccines\u003c/h2\u003e \u003cp\u003eThe biological safety of vaccine formulations is crucial for in vivo administration. On the fourth day after the last immunization, the major organs (ileum, stomach, heart, liver, spleen, lungs, and kidneys) were collected from the immunized mice to assess the tissue pathological toxicity of the vaccine formulation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed, the simultaneous application of magnetic-driven nanorobots and magnetic fields does not lead to inflammation at the inoculation site (the small intestine) and does not cause damage to the gastric tissue. Furthermore, the simultaneous use of magnetic-driven nanorobots and magnetic fields does not cause significant pathological changes in major organs (heart, liver, spleen, lungs, and kidneys), indicating that magnet-driven nanorobots possess good biocompatibility and safety.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.10 Antitumor immunotherapy using magnetic-driven nanorobot vaccines\u003c/h2\u003e \u003cp\u003eOral administration of magnetic-driven nanorobot vaccines can effectively enhance mucosal and cellular immune responses. Therefore, it was necessary to evaluate the in vivo antitumor efficacy of oral vaccination. We subcutaneously injected B16-OVA tumor cells. When the tumor sizes reached approximately 50 mm³, the oral vaccine was administered every four days for a total of three doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea). The tumor growth curves showed that oral administration of OVA or MNC@CaMn/OVA did not significantly inhibit melanoma growth. However, the growth of melanomas in the magnetic-driven nanorobot vaccine group with magnetic field was markedly suppressed. Compared to the control group, the tumor volume in the magnet-driven nanorobot vaccine group with magnetic field was reduced by approximately 69% (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb\u0026amp;c). Photos of tumor-bearing mice are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eg, and the photos of the collected tumors are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed. Compared to the tumors in control mice, the tumor sizes were in the magnet-driven nanorobot vaccine group with magnetic field was obviously reduced. Then, tumors collected from the mice were weighed, and the results are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ee. Compared to the tumors in normal mice (average weight: 1.48 g), the tumor weights in the magnetic-driven nanorobot vaccine group with magnetic field were significantly reduced, averaging just 0.58 g. In addition, the magnetic-driven nanorobot vaccine and magnetic field had no significant impact on the body weight of the mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ef). These findings indicate that oral administration of the magnet-driven nanorobot vaccine under magnetic field effectively enhances antitumor immunotherapy. Certain biochemical indicators in the blood of melanoma-bearing mice (AST, ALT, and LDH) can reflect the efficacy and biosafety of the vaccine formulation[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Normal serum values of ALT, AST and LDH in mice ranged from 10.1–96.5 U/L, 36.3-235.5 U/L and 157.4-899.7 U/L, respectively. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eh-j, the levels of ALT, AST and LDH in normal mice were within the normal range, whereas untreated melanoma-bearing mice had average ALT levels of 105.9 U/L, average AST levels of 1291.7 U/L, and average LDH levels of 5808.7 U/L, all far exceeding the normal range for mice. The average ALT levels in the magnet-driven nanorobot vaccine group with magnetic field were significantly reduced and within the normal range for mice. Moreover, the average AST and LDH levels in the magnet-driven nanorobot vaccine group with magnetic field were significantly lower compared to those in the untreated groups. This suggests that the magnet-driven nanorobot vaccine can reduce tumor size and maintain the normal function of mouse organs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we further investigated the antitumor immune response activated by magnetic-driven nanorobot vaccine with magnetic field. The activation of CD8\u003csup\u003e+\u003c/sup\u003e T cell releases interferons, perforin, and granzymes [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Therefore, assessing the infiltration of cytotoxic CD8\u003csup\u003e+\u003c/sup\u003e T cells in tumor tissue is a critical indicator for evaluating the antitumor immune response. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea, CD8\u003csup\u003e+\u003c/sup\u003e T cell staining of tumor tissue sections showed that the CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration induced by the antigen alone and nanorobot vaccine without magnetic field groups was low in tumor tissues, whereas the magnetic-driven nanorobot vaccine with magnetic field obviously increased the level of CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration in tumor tissues. This may be due to the magnetic field driving the nanorobots to rapidly cross the mucus layer, facilitating the interaction between the antigen and APCs in the lamina propria, enhancing antigen presentation, and aiding in the activation of CD8\u003csup\u003e+\u003c/sup\u003e T cells, which further inhibits tumor growth. Another key phenomenon indicating enhanced tumor immune cycle reactions is the significant activation of CD8\u003csup\u003e+\u003c/sup\u003e T cells in immune organs. Thus, detecting the proportion of CD8\u003csup\u003e+\u003c/sup\u003e T cells in the spleen is very important. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb\u0026amp;c, the magnetic-driven nanorobot vaccine significantly increased the proportion of CD8\u003csup\u003e+\u003c/sup\u003e T cells in the spleen compared to untreated tumor-bearing mice. This is important for enhancing systemic anti-tumor immune responses. Then, another mouse immune tissues - inguinal lymph nodes were collected and shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ed, the sizes of bilateral inguinal lymph nodes obviously increased in the magnetic-driven nanorobot with magnetic field group compared to the control group, which indicates lymphocyte proliferation. All these results reconfirmed that the magnetic-driven nanorobot vaccine was effective in enhancing the anti-tumor immune response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn order to promote the rapid and active crossing of the intestinal mucosal barrier by the vaccine and to prolong its residence time in the intestinal mucosa, we synthesized biodegradable magnetic-driven nanomotors (MNC@CaMn) loaded with antigens and constructed a magnetic-driven nanomotor vaccine delivery platform. Under the precise regulation of the magnetic field, the residence time of the vaccine in the intestines was significantly prolonged and exhibited the characteristics of swarming motility, which could rapidly aggregate and cross the intestinal mucus layer in a targeted manner, greatly facilitating antigen delivery and effectively inducing mucosal and systemic immune responses. The magnetic-driven nanomotor vaccine delivery system constructed in this study provides a new strategy for the development of efficient oral and mucosal vaccines.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eFoundation\u003c/h2\u003e \u003cp\u003eThis work was granted by the National Natural Science Foundation of China (82404556), the National Natural Science Foundation of China (32471525), the Fundamental and Applied Basic Research Project of Guangzhou (SL2023A04J00799), and the General Project of Natural Science Foundation of Guangdong Province (2022A1515010715)\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eL.H.: Methodology, Data curation, Software, Funding acquisition, Writing-original draft. X.S.: Investigation, Administration, Formal analysis, Data curation. Q.Z.: Formal analysis, Investigation. J.L.: Formal analysis, Investigation. W.C.: Formal analysis, Investigation. R.D.:Conceptualization, Supervision, Resources, Funding acquisition. Z.L.: Writing - review \u0026amp; editing, Supervision, Resources, Funding acquisition. Z.G.: Writing - review \u0026amp; editing, Project administration, Supervision, Resources. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVighi G, Marcucci F, Sensi L, Di Cara G, Frati F. Allergy and the gastrointestinal system. Clin Exp Immunol. 2008;153(Suppl 1):3\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S, Liu H, Zhang X, Qian F. Intranasal and oral vaccination with protein-based antigens: advantages, challenges and formulation strategies. 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Science. 1998;282(5386):121\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"mucosal immunization, oral vaccine, magnetic driven nanorobot, swarming motility","lastPublishedDoi":"10.21203/rs.3.rs-5388438/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5388438/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe continuous secretion of mucus by the intestinal mucosa and the intestinal motility combine to limit the absorption of orally administered vaccines. To extend the residence time of vaccines within the gastrointestinal tract and to improve their mucosal transit, we have developed a technology capable of swiftly and actively traversing the intestinal mucus barrier. In this study, we synthesized a biodegradable magnetic driven nanorobot (MNC@CaMn) loaded with antigen and constructed a magnetic driven nanorobot vaccine delivery platform. Under the precise regulation of the magnetic field, the residence time of the vaccines in the intestine was significantly prolonged, and the vaccine exhibited a swarming motility that could rapidly converge and cross the intestinal mucus barrier in a targeted manner, thus greatly facilitating antigen delivery and presentation and significantly activating CD8\u0026thinsp;+\u0026thinsp;T lymphocytes. In addition, the rough surface of the nanorobot ensured stable antigen loading, while the Mn\u003csup\u003e2+\u003c/sup\u003e in the particles was able to stimulate efficient mucosal and systemic immune responses due to its excellent adjuvant effect. The magnetic driven nanorobot vaccine delivery system constructed in this study provides a new strategy for the development of efficient oral and mucosal vaccines.\u003c/p\u003e","manuscriptTitle":"The swarmable magnetic-driven nanorobots for facilitating trans- intestinal mucosal delivery of oral vaccines to enhance mucosal and systemic immune responses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-19 08:35:29","doi":"10.21203/rs.3.rs-5388438/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aa6f88d0-20a1-4dbf-8a2b-2c102244a803","owner":[],"postedDate":"November 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-29T17:08:53+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-19 08:35:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5388438","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5388438","identity":"rs-5388438","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}