Spatial control over catalyst positioning on biodegradable polymeric nanomotors

Scientists over the world are inspired by biological nanomotors and try to mimic these complex structures. In recent years multiple nanomotors have been created for various fields, such as biomedical applications or environmental remediation, which require a different design both in terms of size and shape, as well as material properties. So far, only relatively simple designs for synthetic nanomotors have been reported. Herein, we report an approach to create biodegradable polymeric nanomotors with a multivalent design. PEG-PDLLA (poly(ethylene glycol)-b-poly(D,L-lactide)) stomatocytes with azide handles were created that were selectively reduced on the outside surface by TCEP (tris(2-carboxyethyl)phosphine) functionalized beads. Thereby, two different functional handles were created, both on the inner and outer surface of the stomatocytes, providing spatial control for catalyst positioning. Enzymes were coupled on the inside of the stomatocyte to induce motion in the presence of fuel, while fluorophores and other molecules can be attached on the outside.

was acquired from Click Chemistry Tools. The Alexa Fluor 647-NHS ester and the Pierce immobilized TCEP resin (4% crosslinked on silica beads, effective functional TCEP concentration > 8 mM) were supplied by Thermo Scientific. Dioxane was purchased from Biosolve Chimie. The Amicon Ultrafree centrifugal filters were purchased from Merck-Millipore. Materials for the SDS-PAGE were bought from Bio-Rad, including the Mini-Protean TGX Stain-Free Gels (4-20%, 12 well) and the Precision Plus Unstained marker. All other chemicals, including the NHS functionalized gold particles (20 nm) conjugation kit and the Amplex red enzyme activity assay kit, were supplied by Sigma-Aldrich.
Nuclear Magnetic Resonance (NMR) was measured at 298 K on a Bruker 400 MHz Avance III HD nanobay spectrometer equipped with a 9.4 T Ascend magnet (400 MHz) and BBFO probe. Chemical shifts are given in parts per million (ppm) with respect to tetramethylsilane (TMS, δ 0.00 ppm) as internal standard for 1 H NMR. 1 H spectra were acquired using 48 scans and a relaxation delay of 9 seconds.
2 Dynamic Light Scattering (DLS) measurements were performed on a Malvern Instruments Zetasizer (ZEN 1600), using Zetasizer Software (Malvern Instruments) for analysis of the data. Samples were loaded in Malvern disposable capillary cells. The average of three size measurements with 10 scans of 10 seconds was taken.
Cryogenic Transmission Electron Microscopy (cryo-TEM) pictures were taken on a JEOL TEM 2100 microscope (JEOL Japan). Analysis and processing of the data was performed using Fiji (a free program developed by NIH and available at https://fiji.sc/). Protocol: EM Science TEM grids were glow discharged with a 208 carbon coater (Cressington). On each grid 3 µL of sample was added, blotted and immediately vitrified through freeze plunging into liquid ethane at 100% humidity using an automatic vitrification robot, FEI Vitrobot™ Mark IV (blot time 1 s, blot force 3). Samples were loaded in a 914 High tilt cryoholder (Gatan, Munich, Germany) and inserted into a JEOL Transmission Electron Microscope 2100 (Japan) at 200 kV. Images were taken with a 4096 x 4096 pixel CCD camera (Gatan). The average dimensions and membrane thickness of each sample were obtained from different regions (images) and analyzed with plot profile tools of Fiji.
Confocal imaging was done on a Leica (Wetzlar, Germany) SP8 confocal microscope equipped with a HC PL APO CS2 40x/1.10 WATER immersion objective. The Detector used was HyD (658nm -783nm) Standard mode. Bidirectional scan direction X and a scan speed of 600 Hz was used. The samples were loaded in an Ibidi glass bottom 8 well µ-slide.
UV-VIS Absorbance was recorded on JASCO V-630 UV-Vis spectrophotometer using a 3.5 mL quartz cuvette with a path length of 1.00 cm. , where D is the particle diffusion coefficient, kB the Boltzmann constant, η the viscosity, T the temperature and d the hydrodynamic diameter) to correlate the tracking coordinates obtained from the displacement of the particles with their size (5). In this experiment we analyzed the movement of stomatocytes filled with catalase or catalase and glucose oxidase with addition of three concentrations of fuel.
Protocol: 10 µl of nanomotor solution was diluted in 1 mL PBS buffer (0.05 M, pH 7), containing different amounts of fuel (Video S1 and 2). After injection in the cell, videos of 60 seconds were taken and processed by the NTA2.2 software. By analyzing the video, x and y coordinates of each particle were determined as a function of time intervals. Mean square displacements obtained for 100 frames by averaging over at least 100 particles per sample were plotted versus the time intervals ( Figure S3).

Enzyme activity assay:
The enzyme activity of catalase was determined by an Amplex red hydrogen peroxide assay. In this assay, the Amplex Red reagent competes for hydrogen peroxide to produce the fluorescent product, resorufin. A calibration curve with different concentrations of catalase was made, starting from 0.1 -4 U. The formed fluorescent resorufin was measured by a Tecan Spark M10 plate reader (excitation at 550 nm and Emission at 590 nm). Catalase samples with various ratios of DBCO coupled to the enzyme were tested and compared to the calibration curve for their activity.

Supplementary Tables
Supplementary