Electronic Supporting Information

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1 Electronic Supporting Information Magnetic Field Guided Chemotaxis of imushbots for Targeted Anticancer Therapeutics Tamanna Bhuyan, a Amit Kumar Singh, a Deepanjalee Dutta, a Aynur Unal, b Siddhartha Shankar Ghosh, a,c and Dipankar Bandyopadhyay a,d * a Centre for Nanotechnology, Indian Institute of Technology Guwahati, Assam , India. b Department of Mechanical Engineering, Indian Institute of Technology Guwahati, Assam , India. c Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Assam , India. d Department of Chemical Engineering, Indian Institute of Technology Guwahati, Assam - * dipban@iitg.ernet.in , India. 1. Catalase Assay Figure S1. (A) Enzyme activity of the innate catalase in uncoated Mushbots and imushbots samples for 8.0% (v/v) H 2 O 2 decomposition (B) Variation in catalase enzyme activity with the change in ph values. Figure S1 A shows the specific activity 1 of the mushroom catalase content in uncoated motor for of H 2 O 2 decomposition was around 1.5 U g -1 (1500 U mg -1 ) whereas the same of the imushbots were found to be 1.35 U g -1 (1350 U mg -1 ). The decrease in the catalase activity in imushbots S1

2 accounts for the slight decrease in the rate of peroxide decomposition, as shown later in Figure S4 B. Figure S1 B shows the decrease in catalase enzyme activity with the increase in acidic ph. The highly acidic or basic ph disrupts the tertiary protein structure, leading to inactivation of the catalase enzyme Characterization of magnetite nanoparticles Figure S2. (A) Transmission electron microscopy (TEM) image and (B) XRD pattern of freshly prepared magnetite nanoparticles. The scale bar at the bottom is 50 nm. Figure S2 A shows the TEM image of freshly prepared magnetite nanoparticle (FeONPs). TEM images revealed that the FeONPs were ~ 35 nm or less in size. 5 The XRD pattern (Figure S2 B) showed characteristic diffraction peaks at 2θ = 30.1, 35.5, 43.15, 57.6, 62.6 corresponding to the (220), (311), (400), (511), 440) crystallographic planes of FeONPs (JCPDS ). 6,7 3. FESEM and EDX analysis of micromotors S2

3 Figure S3. (A) Field emission scanning electron microscopy (FESEM) image of an uncoated Mushbot. The scale bar at the bottom is of 1 µm. (B) FESEM image of imushbot in which the FeONPs were observed over the mushroom surface. The scale bar at the bottom is of 1 µm. (C) Energy Dispersive X-ray (EDX) of the uncoated Mushbot. (D) EDX of an imushbot surface shows the Fe peaks. FESEM image Figure S3 A shows the surface of Mushbot before the magnetite nanoparticle (FeONPs) coating. EDX of the uncoated Mushbot shows the presence of mineral content (Figure S3 C). Figure S3 B shows the aggregates of FeONPs of average size ~ nm. The figure confirmed that presence of magnetic coating on the motor surface. The EDX of FeONPs-coated imushbot shows the presence of elemental iron (Fe) peaks from FeONPs (Figure S3 D). 4. Rate constant measurement for H 2 O 2 decomposition Figure S4. Rate constant (k) of 8.0% (v/v) H 2 O 2 decomposition for (A) uncoated Mushbots and (B) imushbots. The rate of decomposition of H 2 O 2 solution by a catalase-rich uncoated Mushbots and imushbots were analyzed by volumetric titration with KMnO 4. 8 The strength of the KMnO 4 was standardized by 0.2 N oxalic acid. Firstly, for Mushbots, a 30 ml of 8.0% (v/v) H 2 O 2 was taken in a beaker and then 10 mg motors (~ 150 µm size) was suspended in the peroxide solution. After every 5 min time interval, a 300 µl aliquot was withdrawn from the beak and added to 10 ml of 0.2 N H 2 SO 4 solution. Following this, the solution was titrated against standard 0.2 N KMnO 4 solutions. The appearance of a permanent faint pink coloration confirmed the end point of the titration. The rate constant (k) for the H 2 O 2 decomposition was estimated. The same S3

4 procedure was repeated for imushbots. In presence of a Mushbots, the rate of decomposition of 8.0% (v/v) peroxide followed first-order kinetics with the rate constant, k = min -1 (Figure S4 A) whereas for imushbots the rate constant was measured to be k = min -1 (Figure S4 B). It is evident from results that the Fe 3 O 4 NPs coating on the motor resulted in slight decrement in the rate of peroxide decomposition. 5. Vibrating sample magnetometry (VSM) measurements Figure S5. Vibrating sample magnetometry (VSM) hysteresis loop of (A) freshly prepared FeONPs and (B) imushbots. The magnetization curve was obtained from the VSM at 25 º C by varying the magnetic field from 15 to 15 kg. The magnetization curve for FeONPs (Figure S5 A) suggested that the FeONPs were soft ferromagnetic in nature with saturation magnetization (M s ) value of ~ emu g -1. Figure S5 B shows the magnetization curve for imushbots having M s value of ~ emu g -1. The presence of non-magnetic mushroom tissue in the motor resulted in decrement of M s value in this case. S4

5 6. Flame Atomic Absorption Spectroscopy (FAAS) Figure S6. Concentration of iron determined by using the standard curve obtained from atomic absorption spectroscopy. The determination of the iron content in FeONPs and imushbots was done by AAS by taking ferrous sulfate salt (FeSO 4 ) as standard (Figure S5). From the plot, it was determined that for 20 ppm of FeONPs and imushbots, the samples has ppm and 3.41 ppm of iron content respectively. 7. CLSM images of untreated HeLa Cells (Control) Figure S7 (A) Bright field and (B) CLSM image of the untreated HeLa cells. No red fluorescence was observed in the CLSM image in the Figure S7. The Bright field and the CLSM images of the untreated HeLa cells do not show the characteristic red auto-fluorescence of DOX molecules due to the absence of DOX in cellular compartment. S5

6 8. Doxorubicin (DOX) loading on the micromotors Figure S8. Variation in concentration of loaded drug DOX with increase in amount of imushbots. The concentration of DOX in the motors was determined by observing the intensity of the characteristic 590 nm fluorescence peak for DOX molecules. The plot was constructed using the binding efficiency protocol as stated in the experimental section of the manuscript and shows the concentration of DOX loading in a definite amount of DOX@iMushbots. From the plot, it was determined that the 1.07 mg ml -1 equivalent of DOX@iMushbots contained mg ml -1 of DOX. 9. MTT Assay of FeONPs, uncoated Mushbots and free DOX molecules Figure S9. MTT assay of HeLa cells treated with (A) FeONPs, (B) uncoated Mushbots and (C) free DOX for 24 h. The values are represented as mean ± SD of results from three individual experiments. S6

7 The Figure S9 A and B revealed marginal anti-cancer activity of uncoated Mushbots (Agaricus bisporus) and free FeONPs respectively, which showed slight toxicity to HeLa cells at concentrations (C) of 0.5 mg ml -1 of uncoated mushbot and FeONPs, as ~ 78-80% of HeLa cells were found to be viable in culture medium. For free DOX (Figure S9 C), more than ~ 60% of HeLa cells were found to be viable at mg ml -1 dosage. 10. Supporting Video details Video S1: The video shows a catalytic uncoated Mushbot immersed in 8.0% (v/v) peroxide solution. The spontaneous generation of oxygen (O 2 ) bubbles occurs due to the catalase-induced decomposition of H 2 O 2 into water and O 2. Since the O 2 bubbles were rapidly and uniformly liberated in all the directions from the motor surface, no net displacement was observed in H 2 O 2 solution. Video S2: The video shows the migration of an imushbot (~ 150 µm size) in 8.0% (v/v) peroxide solution. The motor shows random motion in peroxide medium. Video S3: The video shows the chemotaxis of a ~ 150 µm imushbot in 8.0% (v/v) peroxide bath. The motor moved towards an alkali NaOH source under the influence of ph gradient maintained by a continuous 0.3 M NaOH drip. The ph gradient was tracked by thymol blue indicator, which turned form colorless to blue as the NaOH front moved from its point to introduction (high ph) to the outer periphery of the petridish (low ph). Video S4: The video shows the repulsion of a ~ 150 µm imushbot in 8.0% (v/v) peroxide bath. The motor moved away from an acidic HCl source under the influence of ph gradient maintained by a continuous M HCl drip. The ph gradient was tracked by thymol blue indicator, which turned form colorless to yellow as the HCl front moved from its point to introduction (low ph) to the outer periphery of the petridish (high ph). S7

8 Video S5: The video shows the acid taxis of a ~ 150 µm imushbot in 8.0% (v/v) peroxide bath. The motor moved towards an acidic catalase-hcl source under the influence of ph gradient maintained by a continuous catalase-hcl drip. The ph gradient was tracked by thymol blue indicator, which turned form colorless to yellow as the HCl front moved from its point to introduction (low ph) to the outer periphery of the petridish (high ph). Video S6: The video clip shows the magnetically-guided motion of an imushbot (~ 150 µm) inside a fuel-free water bath. Video S7: The video clip demonstrates a reciprocating chemo-magneto taxis of a ~ 150 µm imushbot. The motor was initially set to a chemotactic motion towards the alkali dripping thread in which the ph gradient was indicated by the thymol blue indicator. Later, the movement of the motor was deviated with the help of an external magnetic field. Video S8: The video clip demonstrates a reciprocating chemo-magneto taxis of a ~ 150 µm imushbot. The motor was initially set to a chemotactic motion towards the catalase-hcl dripping thread in which the ph gradient was indicated by the thymol blue indicator. Later, the movement of the motor was deviated with the help of an external magnetic field. Video S9: The video shows a DOX-loaded imushbot (~ 100 µm) hitting a HeLa cell inside a petridish. The motor was magnetically-guided towards the cell using a bar magnet. REFERENCES (1) Katoch, R. Analytical Techniques in Biochemistry and Molecular Biology, 1 st ed; Springer- Verlag New York: USA, S8

9 (2) Morgulis, S.; Beber, M.; Rabkin, I. Studies on the effect of the catalase reaction ion concentrations. iv. A theory effect at different hydrogen reaction: iii. Temperature on the catalase. J. Biol. Chem. 1926, 68, (3) Ogura, Y. Catalase Activity at High Concentration of Hydrogen Peroxide. Arch. Biochem. Biophys. 1955, 57, (4) Susmitha, S.; Ranganayaki, P.; Vidyamol, K. K.; Vijayaraghavan, R. Purification and characterization of Catalase enzyme from Agaricus bisporus. Int. J. Curr. Microbiol. Appl. Sci. 2013, 2, (5) Mahmed, N.; Heczko, O.; Söderberg, O.; Hannula, S. P. Room Temperature Synthesis of Magnetite (Fe 3-δ O 4 ) Nanoparticles by a Simple Reverse Co-Precipitation Method. IOP Conf. Series: Mater. Sci. and Eng. 2011, 18, (6) Mahadevan, S.; Behera, S. P.; Gnanaprakash, G.; Jayakumar, T.; Philip, J.; Rao, B. P. C. Size distribution of magnetic iron oxide nanoparticles using Warren Averbach XRD analysis. J. Phys. Chem. Solids 2012, 73, (7) Gautam, R. K; Gautam, P.K.; Banerjee, S.; Soni, S.; Singh, S. K.; Chandra, M. Removal of Ni(II) by magnetic nanoparticles. J. Mol. Liq. 2015, 204, (8) Singh, A. K.; Mandal, T. K.; Bandyopadhyay, D. Magnetically guided chemical locomotion of self-propelling paperbots. RSC Adv. 2015, 5, S9