robotics.sciencemag.org/cgi/content/full/5/43/eaba6137/DC1

Supplementary Materials for

Enzyme-powered Janus platelet cell robots for active and targeted drug delivery

Songsong Tang, Fangyu Zhang, Hua Gong, Fanan Wei, Jia Zhuang, Emil Karshalev, Berta Esteban-Fernández de Ávila,

Chuying Huang, Zhidong Zhou, Zhengxing Li, Lu Yin, Haifeng Dong, Ronnie H. Fang, Xueji Zhang*, Liangfang Zhang*, Joseph Wang*

*Corresponding author. Email: josephwang@ucsd.edu (J.W.); zhang@ucsd.edu (L.Z.); zhangxueji@ustb.edu.cn (X.Z.)

Published 10 June 2020, Sci. Robot. 5, eaba6137 (2020)

DOI: 10.1126/scirobotics.aba6137

The PDF file includes:

Fig. S1. Microscopy images of JPL-motors with Cy5-labeled urease on the surface of a well plate before pipetting. Fig. S2. Propulsion performance of PLs in various media containing 100 mM urea. Fig. S3. Propulsion performance of JPL-motors after 30 min at a urea concentration of 100 mM compared with the motion at initial stage. Fig. S4. Characterizations of non-JPLs with Cy5-labeled urease. Fig. S5. Representative flow cytometry histograms of MDA-MB-231 cells incubated for 30 min with DiD-labeled JPL-motors (orange), PLs (cyan), and RBCs (blue). Fig. S6. Microscopy images of DOX-loaded JPL-motors. Fig. S7. Loading amount of JPL-motors and PLs at various DOX initial inputs. Fig. S8. Propulsion performance of JPL-motors with and without DOX loading at a urea concentration of 100 mM. Fig. S9. SEM image of the binding of JPL-motors to E. coli bacteria. Fig. S10. Comparison of the binding affinity of PLs or JPL-motors to E. coli. Fig. S11. Loading amount of JPL-motors and PLs at various Cip initial inputs. Fig. S12. Propulsion performance of JPL-motors with and without Cip loading in the presence of 100 mM urea. Table S1. Quantifications of protein content of PLs, platelets with Janus modification of sulfo-NHS-biotin and streptavidin (JPL-strep), and JPL-motors with the same cell concentration of 5 × 1011 cells/ml.

Other Supplementary Material for this manuscript includes the following: (available at robotics.sciencemag.org/cgi/content/full/5/43/eaba6137/DC1)

Movie S1 (.avi format). Propulsion performance of JPL-motors in various urea concentrations. Movie S2 (.avi format). Propulsion performance of multiple JPL-motors in the presence of 50 mM urea. Movie S3 (.avi format). Propulsion performance of JPL-motors in various media at a urea concentration of 100 mM. Movie S4 (.avi format). Propulsion performance of PLs in various media containing 100 mM urea. Movie S5 (.avi format). Propulsion performance of JPL-motors after 30 min at a urea concentration of 100 mM compared with that at the initial stage. Movie S6 (.avi format). Propulsion performance of non-JPLs at various urea concentrations. Movie S7 (.avi format). Motion comparison of JPL-motors with and without DOX loading at a urea concentration of 100 mM. Movie S8 (.avi format). Motion comparison of JPL-motors with and without Cip loading at a urea concentration of 100 mM.

SUPPLEMENTARY MATERIALS

Supporting Figures:

Fig. S1. Microscopy images of JPL-motors with Cy5-labeled urease on the

surface of a well plate before pipetting. (A) optical, (B) Cy5 channel, and (C) the

overlay of the two channels. (D) size distribution of JPL-motors (n=200).

Protein concentration (mg/ml)

PLs 1.5494 ± 0.0268

JPL-strep 1.5578 ± 0.0186

JPL-motors 1.6197 ± 0.0192

Table S1. Quantifications of protein content of PLs, platelets with Janus

modification of sulfo-NHS-biotin and streptavidin (JPL-strep), and JPL-motors

with the same cell concentration of 5 × 1011 cells/ml. The standard deviation was

calculated from three independent measurements.

Quantification of urease molecules per JPL-motor

The BCA assay was used to quantify the protein content of unmodified platelets (PLs),

platelets with Janus modification of sulfo-NHS-biotin and streptavidin (JPL-strep)

and JPL-motors, as shown in Table S1. Here we used 1 ml sample for quantification.

The number of urease per JPL-motor was calculated by subtracting the protein content

of JPL-motors by JPL-strep. Since the molecular weight of urease (545 KDa) is much

higher than that of sulfo-NHS-biotin (443.43 g/mol), the resulting subtraction yields

the mass of immobilized urease per all counting platelet cells that corresponds to

0.0619 ± 0.0115 mg. The counting cell numbers are 5×1011 cells. Then, the urease

number per JPL-motors has been calculated by the following Equation 1:

𝑁𝑢𝑟𝑒𝑎𝑠𝑒 =𝑚𝑢𝑟𝑒𝑎𝑠𝑒

𝑀𝑢𝑟𝑒𝑎𝑠𝑒×𝑁𝑐𝑒𝑙𝑙𝑠× 6.02 × 1023 (1)

𝑁𝑢𝑟𝑒𝑎𝑠𝑒: the number of modified urease molecules per JPL-motor

𝑚𝑢𝑟𝑒𝑎𝑠𝑒: the mass of modified urease

𝑀𝑢𝑟𝑒𝑎𝑠𝑒: molecular weight of urease

𝑁𝑐𝑒𝑙𝑙𝑠: the counting cell numbers

The urease amount per JPL-motor is thus calculated to be 136 ± 25 molecules.

Fig. S2. Propulsion performance of PLs in various media containing 100 mM

urea. (A) MSD and (B) corresponding effective diffusion coefficients (Deff) (n=15,

mean ± SEM).

Fig. S3. Propulsion performance of JPL-motors after 30 min at a urea

concentration of 100 mM compared with the motion at initial stage. (A) Optical

tracking trajectories (20 s); (B) Mean-squared displacement (MSD) (n = 15, mean ±

SEM).

Fig. S4. Characterizations of non-JPLs with Cy5-labeled urease. (A) optical, (B)

Cy5 channel, and (C) the overlay of the two channels; (D) Representative flow

cytometry histograms of non-JPLs with Cy5-labeled urease (orange) and unmodified

platelets (red).

Fig. S5. Representative flow cytometry histograms of MDA-MB-231 cells

incubated for 30 min with DiD-labeled JPL-motors (orange), PLs (cyan), and

RBCs (blue).

Fig. S6. Microscopy images of DOX-loaded JPL-motors. (A) optical and (B)

Fluorescent channel; (C) the overlay of the two channels.

Fig. S7. Loading amount of JPL-motors and PLs at various DOX initial inputs (n

= 3; mean ± SD).

Fig. S8. Propulsion performance of JPL-motors with and without DOX loading

at a urea concentration of 100 mM. (A) Optical tracking trajectories (20 s); (B)

MSD (n = 15, mean ± SEM).

Fig. S9. SEM image of the binding of JPL-motors to E. coli bacteria (JPL-motors

are pseudocolored in pink, and E. coli bacteria are pseudocolored in purple).

Fig. S10. Comparison of the binding affinity of PLs or JPL-motors to E. coli. (A)

Quantification of the binding percentage to E. coli incubated with PLs and JPL-

motors for 30 min. ns: no statistical significance, t-test. Representative images of the

overlay of optical, DiD and DAPI channels after the incubation of DiD-labeled (B)

PLs or (C) JPL-motors with DAPI-labeled E. coli.

Fig. S11. Loading amount of JPL-motors and PLs at various Cip initial inputs

(n = 3; mean ± SD).

Fig. S12. Propulsion performance of JPL-motors with and without Cip loading

in the presence of 100 mM urea. (A) Optical tracking trajectories (20 s); (B) MSD

(n = 15, mean ± SEM).

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