Drug-loaded Mg-micromotors preparation and characterization

Figure 1a and Supplementary Movie 1 schematically illustrate the preparation steps of the drug-loaded Mg-based micromotors. The cores of the micromotors are made of Mg microparticles with an average size of ~20 µm. In the study, a layer of Mg microparticles was dispersed onto a glass slide, followed by an asymmetrical coating of the microspheres with a thin TiO 2 layer using atomic layer deposition (ALD). The ALD process leads to a TiO 2 uniform coating over the Mg-microspheres, while leaving a small opening (essential for contact with the acid fuel) at the sphere-glass contact point36, which forms a Janus microstructure. Such TiO 2 layer acts as a shell scaffold that maintains the micromotor spherical shape and the opening size during the propulsion, leading to consistent and prolonged operation. The Mg-TiO 2 Janus microparticles were then coated with a PLGA film containing the CLR antibiotic payload. After the drug-loading step, the microparticles were coated with an outer thin chitosan layer (thickness ~100 nm) that ensures efficient electrostatic adhesion of the micromotors to the mucosal layer on the stomach wall while protecting the CLR-loaded PLGA layer. Finally, the resulting CLR-loaded Mg-based micromotors were separated and collected by soft mechanical scratching of the glass slide, leaving a small opening for spontaneous Mg-acid reaction when the motors are placed in an acidic solution. This reaction generates hydrogen microbubbles and leads to efficient propulsion in the stomach fluid24. The small opening enables also a slow reaction process and gradual dissolution of the Mg core, leading to a prolonged micromotor lifetime of ~6 min. The in vivo self-propulsion in the gastric fluid of a stomach and the corresponding drug delivery process from the PLGA layer of the Mg-based micromotors are illustrated schematically in Fig. 1b and Supplementary Movie 2.

Fig. 1 Synthesis and characterization of drug-loaded Mg-based micromotors. a Schematic preparation of the micromotors: Mg microparticles dispersion over a glass slide, TiO 2 atomic layer deposition (ALD) over the Mg microparticles, drug-loaded PLGA deposition over the Mg-TiO 2 microparticles, and Chitosan polymer deposition over the Mg-TiO 2 -PLGA microparticles. b Schematic of in vivo propulsion and drug delivery of the Mg-based micromotors in a mouse stomach. c Time-lapse images (2 min intervals, taken from Supplementary Movie 3) of the propulsion of the drug-loaded Mg-based micromotors in simulated gastric fluid (pH ~1.3). d Schematic dissection of a drug-loaded micromotor consisting of a Mg core, a TiO 2 shell coating, a drug-loaded PLGA layer, and a chitosan layer. e Scanning electron microscopy (SEM) image of a drug-loaded Mg-based micromotor. f, g Energy-dispersive X-ray spectroscopy (EDX) images illustrating the distribution of f magnesium and g titanium in the micromotor. h–k Microscopy images of dye-loaded Mg-based micromotor: h optical image and fluorescence images showing the dye-loaded Mg-based micromotors in the i DiD channel (PLGA layer), j FITC channel (chitosan layer), along with an overlay of the two channels k Full size image

The ability of drug-loaded Mg-based micromotors to efficiently propel in gastric acid was first tested in vitro by using a simulated gastric fluid (pH ~1.3). The microscopic images in Fig. 1c (taken from Supplementary Movie 3 at 2 min intervals) illustrate the fast and prolonged autonomous propulsion of a CLR-loaded Mg-based micromotor in the gastric fluid simulant. The efficient hydrogen bubble generation propels the micromotors rapidly, with an average speed of ~120 μm s−1 (corresponding to a relative speed of 6 body length s−1), and indicates that the Mg-based micromotors can react and move fast in the gastric fluid. Such efficient micromotor propulsion is essential for the motors to reach stomach wall and thus achieving significant therapeutic efficacy. Importantly, the acid-Mg reaction responsible for the autonomous propulsion also spontaneously depletes protons in gastric fluid and thus neutralizes the stomach pH without using PPIs24.

Figure 1d schematically illustrates the structure of a drug-loaded Mg-based micromotor, showing the Mg core, covered mostly with the TiO 2 shell layer, drug-loaded PLGA layer, and an outer chitosan layer. The drug-loaded Mg-based micromotors were carefully characterized. The scanning electron microscopy (SEM) image of a drug-loaded micromotor (shown in Fig. 1e) confirms the presence of a small opening (~2 µm) on the spherical micromotor, produced during the coating process, that exposes the Mg core of the micromotor to the gastric fluid and facilitates the hydrogen bubble thrust. Energy-dispersive X-ray (EDX) spectroscopy mapping analysis was carried out to confirm the motor composition. The resulting EDX images, shown in Fig. 1f and g, illustrate the presence and distribution of magnesium and titanium, respectively.

A fluorescence study was carried out to confirm efficient drug-loading within the PLGA layer, and the coating of the micromotor with the protective and adhesive chitosan layer. This was accomplished by preparing Mg-based micromotors with the PLGA and chitosan coatings containing the fluorescent dyes 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD, λ em = 665 nm), and fluorescein isothiocyanate-dextran (FITC, λ em = 520 nm), respectively. An optical image of a dye-loaded micromotor is displayed in Fig. 1h. The corresponding fluorescence images show the dye-loaded Mg-based micromotor in the DiD and FITC channels (Fig. 1i and j, respectively); an overlay of the two channels is displayed in Fig. 1k. The high-fluorescent intensity of the loaded dyes confirms the successful coating of the micromotor with both PLGA and chitosan layers, along with the high cargo-loading capacity of the micromotor.

Prior to in vivo therapeutic application of the Mg-based micromotors, several in vitro studies were performed. Initially, the ability of drug-loaded micromotors to efficiently propel in gastric acid was tested in vitro. Supplementary Fig. 1a–d displays time-lapse images (corresponding to Supplementary Movie 4) showing the motion of the drug-loaded Mg-based micromotors in simulated gastric fluid adjusted to different pH values (0.75, 1.25, 1.5, and 1.75, respectively). Time-lapse images in Supplementary Fig. 1e–h show the lifetime of a drug-loaded micromotor in gastric fluid simulant (pH ~1.3) to be ~6 min. Supplementary Fig. 1i displays the pH-dependent speed of the micromotor in the gastric fluid simulant. The micromotor speed drastically decreases upon changing the pH of the gastric fluid solution from pH 1.5–1.75. Assuming that the stomach pH is 1.3, the drug-loaded Mg-based micromotors can efficiently move at this condition with an average speed of ~120 μm s−1 (~6 body length s−1).

Drug-loading optimization and in vitro bactericidal activity

The CLR-loading onto the Mg-based micromotors was optimized to achieve a clinically relevant therapeutic concentration of the drug (15–30 mg kg−1 day−1)37. Figure 2a shows a schematic displaying the loading of CLR onto the micromotors. Briefly, the Mg-TiO 2 microparticles dispersed onto a glass slide (~2 mg of Mg microparticles per glass slide) were coated with a PLGA solution prepared in ethyl acetate, which was mixed with CLR (see detailed experimental protocol in “Methods” section). Rapid evaporation under nitrogen current leads to the formation of a homogeneous PLGA-CLR coating over the Mg-TiO 2 microparticles (microscope images of the coated micromotors are displayed in Fig. 2b). The microparticles were further coated with chitosan before quantifying the CLR-loading efficiency of the micromotors. To optimize the drug-loading, Mg-based micromotors were coated with PLGA solutions containing different amounts of CLR (between 4 and 6 mg). By studying different combinations of the PLGA-CLR solution volume and CLR concentration, the highest CLR-loading efficiency (26%), corresponding to 1032 ± 37 µg per 2 mg micromotor, was obtained when coating the microparticles with 120 µL of the PLGA solution containing 4.8 mg of CLR (Fig. 2c, II). This formulation offered optimal CLR-loading and was selected for subsequent in vitro and in vivo anti-H. pylori studies.

Fig. 2 Antibiotic drug loading of the Mg-based micromotors and in vitro bactericidal activity. a Schematic displaying the loading clarithromycin (CLR) onto the Mg-based micromotors. PLGA polymer dissolved in ethyl acetate is mixed with CLR, and the solution is deposited over the Mg-TiO 2 microparticles resulting in the formation of a thin PLGA-CLR coating. b Microscope images showing the PLGA-CLR film over the Mg-based micromotors. Scale bars 100 µm and 40 µm, respectively. c Quantification of CLR-loading amount and yield of the micromotors prepared with different CLR solutions: (I) 100 µL of 40 mg mL−1 CLR solution, (II) 120 µL of 40 mg mL−1 CLR solution, and (III) 200 µL of 30 mg mL−1 CLR solution. All the CLR-loaded Mg-based micromotors were coated with a thin chitosan layer; all samples were dissolved in acid for 24 h before the drug-loading measurement. d In vitro bactericidal activity of free CLR, CLR-loaded Mg-based micromotors, and blank Mg-based micromotors (without CLR drug) against H. pylori bacteria. Error bars estimated as a triple of s.d. (n = 3) Full size image

Once confirmed that the micromotors were capable to load antibiotic cargo with high-loading efficiency, an in vitro bactericidal activity of CLR-loaded Mg-based micromotors against H. pylori was performed. To mimic the gastric environment, samples were treated in 0.1 N HCl for 1 h prior to incubation with bacteria. This also ensured the dissolution of micromotors and consecutive drug release. Figure 2d shows the enumerated amount of bacteria after being treated by CLR-loaded Mg-based micromotors or free CLR solution with varying concentrations of CLR. According to the results, drug-loaded micromotors exhibited a comparable bactericidal activity to free drug solution over the whole range of concentrations used in the study. Specifically, we determined the minimal bactericidal concentration (MBC) values of the samples, defined as the minimal concentration of an antimicrobial agent that kills 3 logs (99.9%) of the bacteria. The MBC value for CLR-loaded Mg-based micromotors was found to be 0.25 μg mL−1, which was unaltered from the MBC value of free CLR. Moreover, bare Mg-based micromotors, with corresponding amount of motors and treated under the same conditions as the free CLR and CLR-loaded Mg-micromotors, were used as negative controls. From Fig. 2d, the bare motors had negligible effect on the viability of H. pylori over the studied range, which supports that the bactericidal effect of CLR-loaded Mg-based micromotors is solely due to the loaded antibiotics, and not due the other compositions of the micromotor carrier or the micromotor acidic environment. Overall, Fig. 2d verifies that the activity of the loaded drug was not compromised compared to free drug. Our in vitro results verified also that drug-loaded micromotors, made of Mg and other degradable materials, eventually destroy themselves and disappear in the acidic environment after releasing the CLR, with no apparent residues in the tissue. The findings validate the potential use of these drug-loaded micromotors for therapeutic applications.

In vivo micromotor retention in mouse stomach

After the optimization of drug-loading onto the Mg-based micromotors and the confirmation of effective in vitro bactericidal activity, the micromotors were further investigated under in vivo setting. First, the in vivo retention properties of the Mg-based micromotors on stomach tissue were examined at different post-administration times, and compared with control groups administered with DI water (Fig. 3). For this purpose, Mg-based micromotors prepared with DiD-labeled PLGA and FITC-labeled chitosan coatings were administered to a group of mice (n = 3), and following 30 min and 2 h of the samples administration, the mice were killed and the entire stomach was excised and opened. Subsequently, the luminal lining was rinsed with PBS and flattened for imaging. Accordingly, Fig. 3a shows bright-field and fluorescence images of the luminal lining of freshly excised mouse stomach at 0 min after oral gavage of DI water, and at 30 min and 2 h after oral gavage of Mg-based micromotors. As can be observed, the images corresponding to the dye-loaded Mg-based micromotors show an intense fluorescent signal in both red and green light channels, which indicates efficient distribution and retention of the micromotors in the mouse stomach. The continuous propulsion of the micromotors and the adhesive properties of the chitosan coating help to achieve a homogeneous distribution of the micromotors in the stomach. The corresponding fluorescence quantification of the dye-loaded micromotors retained in the mouse stomach after 30 min and 2 h oral gavage of the sample is displayed in Fig. 3b. The graphic represents the higher fluorescence signals obtained at 665 and 520 nm (corresponding to DiD and FITC dyes, respectively) for each sample. These results indicate that the micromotors can effectively propel in gastric fluid and are retained in the stomach wall, including the antrum, where the H. pylori bacteria reside. Such highly enhanced retention in the stomach, which is a major advantage of motor-enabled delivery, has been carefully examined in our early studies22,23,24. The powerful propulsion leads to tissue penetration and binding, so that the drug-loaded motor could reach the whole stomach wall for enhanced retention.

Fig. 3 Retention of the Mg-based micromotors in mouse stomachs. a Bright-field and fluorescence images of the luminal lining of freshly excised mouse stomachs at 0 min after oral gavage of deionized (DI) water (control), and at 30 min and 2 h after oral gavage of the Mg-based micromotors. Scale bar 500 mm. b Corresponding fluorescence quantification of all the images shown in a. Error bars estimated as a triple of s.d. (n = 3) Full size image

In vivo anti-H. pylori therapeutic efficacy

We proceeded to test the in vivo therapeutic efficacy of the drug-loaded Mg-based micromotors against H. pylori infection. Prior to the therapeutic study, we developed H. pylori infection in a mouse model using C57BL/6 mice. Each mouse was inoculated with 3 × 108 CFU H. pylori SS1 in brain–heart infusion (BHI) broth by oral gavage three times on day 3, 5, and 7 (Fig. 4a)38, 39. Two weeks after inoculation, the H. pylori-infected mice were divided into five groups (n = 6, for each group) and orally administered with DI water, blank Mg-based micromotors (without CLR drug), free CLR drug with PPI (CLR+PPI), CLR-loaded silica microparticles, or CLR-loaded Mg-based micromotors once a day for five consecutive days. On each day of treatment, mice in the free CLR+PPI group received 400 µmol kg−1 of omeprazole (as PPI treatment) 30 min before administrating CLR, to neutralize gastric acid and prevent potential degradation of CLR. Such PPI dosage has been reported to be effective both in reducing the gastric acidity in mouse models40, as well as in preserving the effectiveness of co-administered antibiotics39, 41, 42. After the treatment course, the bacterial burden was evaluated by enumerating and comparing H. pylori counts recovered from each mouse stomach. The mean bacterial burden from two negative control groups treated with DI water and blank Mg-based motors were 2.1 × 107 and 1.4 × 107 CFU g−1 of stomach tissue, respectively (Fig. 4b, black and orange color, respectively). Meanwhile, a bacterial burden of 3 × 106 CFU g−1 was measured from the mice treated with CLR-loaded silica microparticles, which did not show statistical difference to the negative controls. In contrast, when the mice were treated with CLR-loaded Mg-based micromotors, the bacterial burden was quantified as 2.9 × 105 CFU g−1, a significant reduction compared with the negative control and CLR-loaded silica microparticle groups. The substantial improvement in H. pylori reduction demonstrates the benefit of acid-powered Mg-based micromotors compared with static micron-sized carriers. A bacterial burden of 2.8 × 106 CFU g−1 was obtained for the positive control mice with free CLR+PPI treatment. Although the difference between CLR-loaded Mg-based micromotors and the free CLR+PPI groups was not statistically significant, the CLR-loaded micromotors reduced the H. pylori burden in mice compared with in the negative controls by ~1.8 orders of magnitude, whereas the free CLR+PPI group reduced it only by ~0.8 orders of magnitude. These results might be derived from the benefit of the propulsion-enabled active drug delivery performed by the Mg-based micromotors in the stomach. These results demonstrate that the Mg-based micromotors can effectively propel and distribute throughout the stomach of living mice to significantly reduce H. pylori levels.

Fig. 4 In vivo anti-H. pylori therapeutic efficacy. a The study protocol including H. pylori inoculation and infection development in C57BL/6 mice, followed by the treatments. b Quantification of bacterial burden in the stomach of H. pylori-infected mice treated with DI water (black color), bare Mg-based micromotors (orange color), free CLR+PPI (green color), CLR-loaded silica microparticles (blue color), and CLR-loaded Mg-based micromotors (red color), respectively (n = 6 per group). Bars represent median values. *P < 0.05, **P < 0.01, ns no statistical significance Full size image

In vivo toxicity evaluation of Mg-based micromotors

Finally, the toxicity profile of the Mg-based micromotors in the stomach as well as in the lower GI tract was evaluated. Healthy mice were orally administered with Mg-based micromotors or DI water once daily for five consecutive days. Throughout the treatment, no signs of distress such as squinting of eyes, hunched posture, unkempt fur, or lethargy were observed in both groups. Initially, the toxicity profile of the Mg-micromotors in the mouse was evaluated through changes in body weight. During the experimental period, mice administered Mg-micromotors maintained a constant body weight compared with the mice administered DI water (Fig. 5a). On day 6, mice were killed and their stomachs and lower GI sections were processed for histological staining. Longitudinal sections of the glandular stomach (Fig. 5b), three major segments of small intestine (duodenum, jejunum, and ileum, Fig. 5c–e, respectively) and the two major segments of large intestine (proximal and distal colon, Fig. 5f–g, respectively) were stained with hematoxylin and eosin (H&E). The stomach and lower GI sections of the micromotor-treated group showed undamaged structure of columnar epithelial cells with no signs of superficial degeneration or erosion (Fig. 5b–g, left). There was no noticeable difference in the gastric and intestinal mucosal integrity, in terms of thickness as well as size and number of crypt and villus, between the motor-treated and DI water-treated groups (Fig. 5b–g, left vs. right part). No lymphocytic infiltration into the mucosa and submucosa was observed, indicating no sign of gastric inflammation. The in vivo toxicity studies of Mg-based micromotors showed no effect on the mouse body weight, apparent alteration of GI histopathology or observable inflammation, suggesting that the treatment of Mg-based micromotors is safe in the mouse model.