Enzymatically active biomimetic micropropellers for the penetration of mucin gels

Abstract

In the body, mucus provides an important defense mechanism by limiting the penetration of pathogens. It is therefore also a major obstacle for the efficient delivery of particle-based drug carriers. The acidic stomach lining in particular is difficult to overcome because mucin glycoproteins form viscoelastic gels under acidic conditions. The bacterium Helicobacter pylori has developed a strategy to overcome the mucus barrier by producing the enzyme urease, which locally raises the pH and consequently liquefies the mucus. This allows the bacteria to swim through mucus and to reach the epithelial surface. We present an artificial system of reactive magnetic micropropellers that mimic this strategy to move through gastric mucin gels by making use of surface-immobilized urease. The results demonstrate the validity of this biomimetic approach to penetrate biological gels, and show that externally propelled microstructures can actively and reversibly manipulate the physical state of their surroundings, suggesting that such particles could potentially penetrate native mucus.

Keywords: Micropropellers; biomimetic penetration of biological gels; gastric mucus barrier; gel-sol transition; helicobacter pylori swimming mechanism; muco-adhesion; propulsion in viscoelastic media; urease surface immobilization.

Fig. 1. Mechanism for mucin penetration.

Schematic illustration of the propulsion strategy of H. pylori through mucin gels and the catalytically active magnetic micropropellers presented here. The enzyme on the helices’ surface hydrolyzes urea and liquefies the environment via the resulting local rise in pH.

Fig. 2. Acid-stable, enzyme-functionalized micropropellers.

(A) Fabrication process of acid-stable, enzyme-functionalized magnetic micropropellers consisting of GLAD on top of a monolayer of silica beads, ALD to protect the resulting magnetic helices, enzyme immobilization, and ultrasonication in solution to remove the particles from the wafer. (B) ESB-SEM (energy-selective backscatter–scanning electron microscopy) image of a magnetic micropropeller, with the Ni section clearly visible. Scale bar, 500 nm.

Fig. 3. Urease activity.

SiO₂ beads (~50 μm in diameter) functionalized with urease using GA coupling, in a solution of 100 mM urea colored with the pH indicator bromothymol blue. The beads were dried on a coverslip and micrographs were recorded 1, 2, 3, 4, and 5 min after the addition of urea solution. The blue coloring clearly demonstrates the increase in pH due to catalytic urea hydrolysis.

Fig. 4. Mucin rheology.

(A) Viscoelastic properties of reconstituted PGMs at different pH conditions. A 2% solution of PGMs exhibits a clear sol-gel transition between pH 4.5 and pH 7. Closed symbols denote the storage modulus G′(f), and open symbols denote the loss modulus G″(f). (B) Gelation of the PGM solution is neither suppressed by the addition of 1 mM bile salts (BS; sodium glycodeoxycholate and sodium taurocholate) nor suppressed by the addition of 20 mM urea, the use of 5.5 mM HCl instead of a pH 4.5 reconstitution buffer, or ultrasonication.

Fig. 5. Propulsion in mucin gels.

(A to D) Tracks of micropropellers with (B and D) and without (A and C) urease immobilized on the surface, in an acidified 2% mucin gel with (A and B) and without (C and D) 20 mM urea over a time frame of 25 s. The particles were propelled in the x direction at a frequency of 30 Hz and a magnetic field strength of 10 mT. Each graph shows the tracks from one video recording. (E) The average velocity under these conditions is shown and represents an average of a minimum of 50 particles tracked over at least 10 s. Only the x component of the velocity was taken into account; the small drift in the y direction that can sometimes be observed when the particles are close to the glass surface [as in (D)], or “kinks” in the y direction due to hydrodynamic interactions with another propeller in proximity [as in the topmost trajectory in (B)], was neglected. The corresponding videos can be found in the Supplementary Materials.