Self-Propelling Targeted Magneto-Nanobots for Deep Tumor

Penetration and pH-Responsive Intracellular Drug Delivery

Presented by: Tejashree

Jayatkar
ARTICLE HISTORY
Received: 20 Accepted: 24 Published online: Guided by: Mr. Gadhwe
December 2019 February 2020 13 March 2020
M. Pharm
(Pharmaceutical Quality
Assurance)

30 September 2021
SINHGAD COLLEGE OF PHARMACY, VADGAON (BK), PUNE
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 Journal name Scientific reports
Impact factor 4.379
Cite score 7.2
Average citations In 2020- 5,40,000

This article was submitted to scientific reports of the journal

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nature Scientific Reports | (2020) 10:4703 |

https://doi.org/10.1038/s41598-020-61586-y 1
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 Reason behind the
selection of the paper:

• To study the various features of

nanotechnology assisted nanobots in
cancer therapy
• To study the multicomponent nanobot’s
design which represents method in
targeting tumors with self assisted anti-
cancer drug delivery.
• Developmental method and parametric
evaluation study of CNT loaded DOX
reduce tumor spheroids more efficiently
than free DOX
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• To study efficacy of nanobots assisted

drug delivery system

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 Introduction

• Miniature robotic system provides

considerable benefits over conventional
& micro/nanoparticle based therapies
• Surface functionalization with a specific
ligand that allows nanocarriers to
increase active targeting ability
• Therefore, Self propelling magnetic
nanobots capable of intrinsic navigation
with enhanced pharmacokinetics &
deeper tissue penetration
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 Multicomponent conjugation
Fe3O4
• Decomposition of H2O2 existing in the tumor
microenvironment TME into water and
oxygen
• Tumor cells produce H2O2 rate 0.5nmol/10^4
cells/h
• Impart autonomous propulsion ability +
superparamagnetic property to nanobot
system
• Impart mechanism of ‘on demand’ release
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Multiwalled CNT
• CNT offers chemical tunability
• Transformed passive CNTs into active
autonomous nano-propelled-bots with
controlled anti-cancer drug delivery platform
Nanobots designed by chemical coordination
Fe3O4 NPs + anti –EpCAM mAb to CNT + reactive
spacer glutathione (GSH) + anticancer drug DOX
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 • Carbon nano tube

Materials & methods

a) Multiwalled carbon nanotubes
b) (Outer diameter 10-15µm, length 1-5µm
purity > 99%)
c) Ferric chloride tetrahydrate, Ferrous
chloride tetrahydrate
d) Transferrin Tf
e) EDC HCl
f) Glutathione
g) Doxorubicin hydrochloride
h) HCT 116 cells
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 Functionalization of CNTs & synthesis of Fe3O4 GSH

Fe3O4 NPs- Coprecipitation of ferric+

CNT purified and oxidized
ferrous ions (2:1) heat at 80°C for 30
min in aqs ammonium hydroxide soln

85 mg of CNT dispersed in 100 ml Wash with H20+ethanol then 5mg of

Fe3O4 NPs dispersed in 150µl of H2O
& 50µl of methanol, sonicate for 15
min
Mixture of H2SO4/HNO3(3:1),
sonicate for 6h

4mg GSH dissolve in 50µl of H20, add

in above soln sonicate for 2h
Mixture diluted with 100 ml ice
cool H2O
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Concentrated by centrifugation & GSH functionalized NPs were then

washed with 5% NaOH soln, pure isolated by magnetic separation, wash
H2O then dry functionalized CNT with H2O & dried well
at 80°C for 12h
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 Loading of DOX in CNT-COOH Synthesis of CNT-DOX Fe3O4 Synthesis of CNT-DOX-Fe3O4 Tf

20 mg of CNT-COOH 20 mg of CNT-DOX+ 5mg of 1o mg of CNT-DOX-

suspended in 5ml solution of EDC + 5 ml of phosphate Fe3O4 in 1ml of EDC
DOX (8mg/ml) buffer, agitated for 30 min solution

Add 1 ml of Tf
Solution sonicated for 6h & 20 mg of Fe3O4-GSH solution (5mg/ml),
allowed to stand for 12h agitated for 1h reaction agitated for
4h

Synthesized product, CNT-DOX Conjugated CNT-DOX-Fe3O4 Synthesized NPs

collected by centrifugation & NPs were magnetically collected by magnetic
dried well at room temp separated and dried at 40°C separation
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 Antibody/Tf targeted nanobot conjugation & characterization
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Presence of Fe3O4 NPs at the tip

Fe3O4 NPs analyzed by HRTEM in SAED confirming crystalline Fe3O4
ends of CNTs
magnetic crystalline nature structure
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 • Characterization by FTIR
• • Zeta potential
CNT peak at 1715 cm−1
• • CNT-COOH - 4.07 mV
DOX loaded CNT at- peak of DOX- 998 cm−1
• • Fe3O4 - 18.6 mV
Peak of DOX with CNT 1213 cm−1
• • CNT-DOX-Fe3O4 - 8.9 mV
CNT-DOX-Fe3O4- 575 cm−1, 629 cm−1
• CNT-DOX-Fe3O4-Tf - 22.2 mV
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• CNT-DOX-Fe3O4-Tf- 3448 cm−1, 1645 cm−1

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UV-visible spectra of DOX (λmax=480nm), Tf Normalized fluorescence spectra of

(λmax=280nm) and CNT-DOX-Fe3O4-Tf (Tf peak at DOX and CNT-DOX-Fe3O4-Tf (λex=480nm, λem
280nm and DOX peak at 480nm) =590nm
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 Motion & position kinetic analysis of nanobots

Different biological fluid medium Time-lapse images of the nanobot driven by

PBS- Phosphate buffer saline (pH 7.4) oxygen bubble propulsion after time intervals of
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DMEM- Dulbecco’s modified eagle (a) 0, (b) 2.0, 4.6, 6.5 and 10 s. Speed of
medium- cell media nanobot in the presence of different
Blood serum concentration of H2O2 (0.5–8w/v %)
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(C) PBS, (D) DMEM and (E) serum, (F) Analysis of force of nanobot in PBS, DMEM and serum in
presence of diferent concentration of H2O2 (0.5–8w/v %).
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 Drug release profiles of nanobots

Fluorescent images of HCT116 cells treated with free DOX, CNT-DOX-Fe3O4 and CNT-DOXFe3O4-Tf. (A) At 4h
exposure and at pH 7.4, DOX released from CNT-DOX-Fe3O4 and CNT-DOX-Fe3O4-Tf was observed to be localized in
the nuclear region (A,B). The intracellular release of DOX can be attributed to the opening of pH-sensitive nano gates
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due to amide bond cleavage in the acidic lysosomal compartments. Cells incubated with free DOX showed efflux of
DOX from the nucleus back into the cytoplasm, which is in contrast to the findings for CNT-DOX-Fe3O4 and CNT-DOX-
Fe3O4-Tf. (B) At 4h exposure and at pH 6.5, the fluorescence intensity of DOX from CNT-DOX-Fe3O4-Tf nanobot was
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 At 24h and at pH 7.4, most of the DOX was released from CNT-DOX-Fe3O4-Tf suggesting the efficient release of
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DOX from interior cavity of CNT after opening of Fe3O4 nano gate in lysosomal conditions. (D) At 24h and at pH
6.5, the fluorescence intensity of DOX in the cells was more pronounced suggesting enhanced cellular
internalization of CNT-DOX-Fe3O4-Tf nanobot
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 Kinetic study of Fe3O4 NP uncapping and DOX release from CNT-DOX-Fe3O4-Cy5-Tf nanobots in cells using confocal
microscopy. Time-dependant release of DOX (red) into the acidic lysosomal compartment (green, LysoTracker) over
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4h, indicating -cleavage of CNT- Fe3O4 amide-bond, subsequent

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 Antitumor efficacy of drug loaded nanobots on 3D spheroidal tumors

Anti-tumor efficacy of administered nanobots on HCT116

spheroids. (A) The tumor mass is expressed as area over a
period of 72h. Tumor disintegration by reduction in tumor area
of spheroids treated with CNT-DOX-Fe3O4-mAb and CNT-DOX-
Fe3O4-Tf. (B) Tumor viability under various treatments are
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depicted as percent survival, compared to control.

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Nanobot’s efficacy as a drug delivery vehicle

• Cytotoxicity analysis of all this nanobots

incubated for 48h with HCT116 cells.
• Cell viability study of treatments with free
DOX and nanobots reveals a statistical
improvement of CNT-DOX-Fe3O4-Tf and
CNT-DOX-Fe3O4-mAb nanobots over free
DOX treatment of HCT116 cells.
• The CNT-Fe3O4 nanobot does not show
greater cytotoxic effect as compared to
the control-CNT treatment.
• In contrast, the CNT-DOX-Fe3O4-Tf and
CNT-DOX-Fe3O4-mAb nanobot loaded
with an equivalent dose of DOX shows
statistically significant improvement in the
toxicity induced, suggesting greater
efficacy of the DOX delivery by nanobot.

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 Conclusion

• Demonstration of a novel self-powered multifunctional gated nanobot that offers promising

alternative drug delivery system based on rapid autonomous motion for quicker and deeper
delivery to the tumor site.
• This nanobot system combines several intriguing features, namely self-propulsion, high DOX
loading, tumor targeting and profound penetration ability, in situ pH triggered release of the DOX,
and improved drug availability.
• The CNT-DOX-Fe3O4-Tf nanobots demonstrated ultrafast self-propulsion (0.972 and 0.535 mm
s−1 ) not only in high ionic media (PBS buffer) but also in biological media such as DMEM (2.333
and 1.120mms−1 ) and blood serum (8.026 and 1.120mms−1 ), a crucial ability necessary for its
use in biomedical applications.
• The cellular uptake study showed controlled release of DOX due to opening of pH-sensitive nano
gates by cleavage of amide bond in the acidic lysosomal compartments
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THANK YOU

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