Gastrointestinal tract drug delivery using algae motors embedded in a degradable capsule

Abstract

The use of micromotors for active drug delivery via oral administration has recently gained considerable interest. However, efficient motor-assisted delivery into the gastrointestinal (GI) tract remains challenging, owing to the short propulsion lifetime of currently used micromotor platforms. Here, we report on an efficient algae-based motor platform, which takes advantage of the fast and long-lasting swimming behavior of natural microalgae in intestinal fluid to prolong local retention within the GI tract. Fluorescent dye or cell membrane-coated nanoparticle functionalized algae motors were further embedded inside a pH-sensitive capsule to enhance delivery to the small intestines. In vitro, the algae motors displayed a constant motion behavior in simulated intestinal fluid after 12 hours of continuous operation. When orally administered in vivo into mice, the algae motors substantially improved GI distribution of the dye payload compared with traditional magnesium-based micromotors, which are limited by short propulsion lifetimes, and they also enhanced retention of a model chemotherapeutic payload in the GI tract compared with a passive nanoparticle formulation. Overall, combining the efficient motion and extended lifetime of natural algae-based motors with the protective capabilities of oral capsules results in a promising micromotor platform capable of achieving greatly improved cargo delivery in GI tissue for practical biomedical applications.

Fig. 1.. Schematic of algae motors in a capsule for gastrointestinal tract delivery.

(A) Algae motors loaded within protective capsules containing an inner hydrophobic coating layer and an outer enteric coating layer can be used for oral delivery applications. (B) The algae motor-loaded capsule first enters the stomach, where the enteric coating protects it from degradation at acidic gastric pH. Upon entering the intestines, the enteric coating is dissolved in the nearly neutral pH and the capsule is degraded, leading to complete release of the algae motors. (C) Brightfield (upper) and fluorescent (lower) images of an algae motor-loaded capsule. Scale bar, 2 mm. (D) Representative tracking trajectories demonstrating the autonomous movement of algae motors in simulated intestinal fluid. (E) Representative biodistribution of fluorescently labeled algae motors in the GI tract 5 h after administration in a capsule by oral gavage.

Fig. 2.. Motility of algae motors and magnesium motors in simulated intestinal fluid (SIF) at room temperature.

(A) Speed of algae motors and Mg motors in SIF during 12 h of operation (n = 5, mean ± SD). (B) The percentage of motile motors in SIF during 12 h period (n = 5, mean + SD). (C,D) Time-lapse snapshots and trajectories of algae motors (C) and Mg motors (D) over a span of 2 s at different timepoints during operation. Scale bars, 50 μm. (E-J) Time-lapse images showing trajectories over a span of 1 s and corresponding speed distributions of unmodified algae (E,F), fluorescein-labeled algae motors (G,H), and algae motors carrying DiI-loaded RBC membrane-coated nanoparticles (I, J) (n = 100). Scale bars, 20 μm.

Fig. 3.. Loading and release of algae motors in a capsule in vitro.

(A) Brightfield and fluorescence microscopy images of autofluorescence of algae motors (red) in a capsule formulation fabricated with a DiO-labeled octadecyltrimethoxysilane (OTMS) inner coating (green) and a Pacific Blue-labeled enteric outer coating (blue). Scale bar, 1 mm. (B) Release profile of algae motors from a capsule in simulated gastric fluid (SGF) (blue line) and simulated intestinal fluid (SIF) (red line). Inset images correspond to t = 15 min (left) and t = 45 min (right). (C) Quantification of algae release from capsules over time in SIF (n = 3, mean + SD). (D) Time-lapse images (t = 15 min, 30 min, and 45 min) showing the release of algae motors from a capsule in SIF. Scale bar, 100 μm. (E) Representative tracking lines (captured from movie S7) of encapsulated algae motors in TAP medium and released algae motors in SIF. Scale bar, 50 μm. (F,G) Speed distribution of the encapsulated algae motors (F) and released algae motors (G) from (E) (n = 100).

Fig. 4.. Comparison of the distribution of algae motors and magnesium motors in the gastrointestinal tract.

(A) Speed of algae motors and Mg motors at 37 °C in SIF during 12 h of operation (n = 5, mean ± SD). (B) Percentage of motile algae motors (red line) and Mg motors (blue line) at 37 °C in SIF during 12 h of operation (n = 5, mean ± SD). (C) Representative images of the GI tract of mice 5 h after oral administration of fluorescein-labeled algae motors in a capsule (left) or Mg motors in a capsule (right). (D) Quantitative analysis of total fluorescence intensity within the small intestine from the images in (C) (n = 3, mean + SD). Student’s two-tailed t-test, **p < 0.01.

Fig. 5.. Gastrointestinal tract delivery of algae motors in comparison with other algae control.

(A) Representative ex vivo fluorescence images of GI tissues of mice 5 h after oral administration with TAP medium as negative control, free algae without capsule, static algae in a capsule, and algae motors in a capsule. (B) Quantitative analysis of the mean fluorescence from the experiment in (A) (n = 3, mean + SD). One-way ANOVA, ***p < 0.001, ****p < 0.0001.

Fig. 6.. Characterization of drug-loaded algae motors.

(A) Schematic illustration of the fabrication process for algae-NP(Dox) motor. (B) Brightfield, autofluorescence of algae chloroplast (red), DiO-labeled RBC membrane (green), and merged microscopy images showing the loading of Dox-loaded NP with dye-labeled RBC membrane onto an algae motor. Scale bar: 5 μm. (C) SEM image of an algae motor loaded with NP(Dox). Scale bar, 2.5 μm. Inset shows a zoomed in view corresponding to the dashed red box. Scale bar, 200 nm. (D) Quantification of drug loading amount and loading efficiency of 1 × 10⁶ algae-NP(Dox) at different Dox inputs (n = 3, mean ± SD). (E,F) Mean (E) and median (F) speed of algae-NP(Dox) motor and bare algae. The speed was measured from 100 individual alga. (G) The cumulative drug release profiles from algae-NP(Dox) motor and NP(Dox) (n = 3, mean ± SD). (H) Viability of B16-F10 cancer cell lines after 24 h, 48 h and 72 h of incubation with blank solution, bare algae, NP(Dox), and algae-NP(Dox) motor (n = 3, mean ± SD). (I) Quantification of the total Dox content per small intestine at different times after administration of algae-NP(Dox) motor and NP(Dox) in a capsule (n = 3, mean ± SD). Student’s multiple t-test, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 7.. In vivo safety analysis of algae motors following oral administration.

(A,B) Comprehensive blood chemistry panel (A) and blood cell counting (B) taken from nontreated mice, mice with single dose treatment, and mice with multiple dose treatment (n = 3, mean + SD). For single dose evaluation, mice were orally administrated with one algae motor capsule at day 0, and blood samples were collected at day 1. For multiple doses evaluation mice were orally administrated with one algae motor capsule every other day on days 0, 2, 4, and 6. Blood samples were collected at day 7. The green dashed lines indicate mouse reference ranges of each analyte. (C) Representative H&E-stained histological sections from different sections of the GI tract from nontreated mice and mice treated with the algae motors in a capsule 24 h after oral administration. Scale bar, 100 μm. (D) H&E-stained histological sections of major organs, including the heart, lungs, liver, kidneys, and spleen, from nontreated mice and mice treated with the algae motors in a capsule 24 h after oral administration. Scale bar, 250 μm.