The Photothermal Effect of Targeted Methotrexate-Functionalized Multi-Walled Carbon Nanotubes on MCF7 Cells

Document Type : Research article


1 Department of Pharmaceutics, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

2 Department of Pharmaceutics, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran

3 Department of Medicinal Chemistry, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

4 Department of Toxicology and Pharmacology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.


Purpose: our goal is to reduce the release rate of methotrexate (MTX) and increase cell death efficiency.
Methods: Carboxylated multi-walled carbon nanotubes (MWCNT-COOH) were functionalized with MTX as a cytotoxic agent, FA as a targeting moiety and polyethylene amine (PEI) as a hydrophilic agent. Ultimately, MWCNT-MTX and MWCNT-MTX-PEI-FA were synthesized. Methotrexate release studies were conducted in PBS and cytotoxic studies were carried out by means of the MTT tassay.
Results: Methotrexate release studies from these two carriers demonstrated that the attachment of PEI-FA onto MWCNT-MTX reduces the release rate of methotrexate. The IC50 of MWCNT-MTX-PEI-FA and MWCNT-MTX have been calculated as follows: 9.89 ± 0.38 and 16.98 ± 1.07 µg/ml, respectively. Cytotoxic studies on MWCNT-MTX-PEI-FA and MWCNT-MTX in the presence of an IR laser showed that at high concentrations, they had similar toxicities due to the MWCNT’s photothermal effect. Targeting effect studies in the presence of the IR laser on the cancer cells have shown that MWCNT-MTX-PEI-FA, MWCNT-MTX and f-MWCNT have triggered the death of cancer cells by 55.11±1.97 %, 49.64±2.44 %, and 37±0.70 % respectively.
Conclusions: The release profile of MTX in MWCNT-MTX-PEI-FA showed that the presence of PEI acts as a barrier against release and reduces the MTX release rate. In the absence of a laser, MWCNT-MTX-PEI-FA exhibits the highest degree of cytotoxicity. In the presence of a laser, the cytotoxicity of MWCNT-MTX and MWCNT-MTX-PEI-FA has no significant difference. Targeting studies have shown that MWCNT-MTX-PEI-FA can be absorbed by cancer cells exclusively.


Main Subjects

(1) Chan E and Cronstein BN. Mechanisms of action of
methotrexate. Bull. Hosp. Jt. Dis. (2013) 71: S5-S8.
(2) Tian H and Cronstein BN. Understanding the
mechanisms of action of methotrexate. Bull. NYU
Hosp. Jt. Dis. (2007) 65: 168-73.
(3) Sykes EA, Chen J, Zheng G and Chan WC.
Investigating the impact of nanoparticle size on
active and passive tumor targeting efficiency. ACS
nano 8 (2014) 6: 5696-5706.
(4) Lammers T, Kiessling F, Hennink WE and Storm
G. Drug targeting to tumors: principles, pitfalls and
(pre-) clinical progress. J. Control. Release. (2012)
161: 175-87.
(5) Wang L, Su W, Liu Z, Zhou M, Chen S, Chen Y,
Lu D, Liu Y, Fan Y and Zheng Y. CD44 antibodytargeted liposomal nanoparticles for molecular
imaging and therapy of hepatocellular carcinoma.
Biomaterials (2012) 33: 5107-14.
(6) Kumar A, Ma H, Zhang X, Huang K, Jin S, Liu J, Wei
T, Cao W, Zou G and Liang X-J. Gold nanoparticles
functionalized with therapeutic and targeted peptides
for cancer treatment. Biomaterials (2012) 33: 1180-
(7) Das M, Datir SR, Singh RP and Jain S. Augmented
Anticancer Activity of a Targeted, Intracellularly
Activatable, Theranostic Nanomedicine Based on
Fluorescent and Radiolabeled, Methotrexate-Folic
Acid-Multiwalled Carbon Nanotube Conjugate.
Mol. Pharm. (2013) 10: 2543-57.
(8) Wilczewska AZ, Niemirowicz K, Markiewicz KH
and Car H. Nanoparticles as drug delivery systems.
Pharmacol Rep. (2012) 64: 1020-37.
(9) Luo Y, Wang S, Shen M, Qi R, Fang Y, Guo R, Cai
H, Cao X, Tomás H and Zhu M. Carbon nanotubeincorporated multilayered cellulose acetate
nanofibers for tissue engineering applications.
Carbohydr Polym. (2013) 91: 419-27.
(10) Venkatesan J, Ryu B, Sudha P and Kim S-K.
Preparation and characterization of chitosan–
carbon nanotube scaffolds for bone tissue
engineering. Int. J. Biol. macromol. (2012) 50:
(11) Okamoto M and John B. Synthetic biopolymer
nanocomposites for tissue engineering scaffolds.
Prog. Polym. Sci. (2013) 38: 1487-503.
(12) Eatemadi A, Daraee H, Karimkhanloo H, Kouhi M,
Zarghami N, Akbarzadeh A, Abasi M, Hanifehpour
Y and Joo SW. Carbon nanotubes: properties,
synthesis, purification, and medical applications.
Nanoscale Res. lett. (2014) 9: 393-.
(13) He H, Pham-Huy LA, Dramou P, Xiao D, Zuo P
and Pham-Huy C. Carbon nanotubes: applications
in pharmacy and medicine. BioMed Res. Int. (2013)
(14) Wujcik EK and Monty CN. Nanotechnology
for implantable sensors: carbon nanotubes and
graphene in medicine. Wiley Interdiscip Rev:
Nanomed Nanobiotechnol. (2013) 5: 233-49.
(15) Bilalis P, Katsigiannopoulos D, Avgeropoulos A
and Sakellariou G. Non-covalent functionalization
of carbon nanotubes with polymers. RSC Adv.
(2014) 4: 2911-34.
(16) Tian Z, Shi Y, Yin M, Shen H and Jia N.
Functionalized multiwalled carbon nanotubesanticancer drug carriers: synthesis, targeting ability
and antitumor activity. Nano Biomed Eng. (2011)
(17) Wang S, Ng CW, Wang W, Li Q and Hao Z.
Synergistic and competitive adsorption of organic
dyes on multiwalled carbon nanotubes. Chem. Eng.
J. (2012) 197: 34-40.
(18) Yang Z, Wang Z, Tian X, Xiu P and Zhou R. Amino
acid analogues bind to carbon nanotube via π-π
interactions: comparison of molecular mechanical
and quantum mechanical calculations. J. Chem.
Phys. (2012) 136: 01B607.
(19) Liu X, Tao H, Yang K, Zhang S, Lee S-T and Liu Z.
Optimization of surface chemistry on single-walled
carbon nanotubes for in-vivo photothermal ablation
of tumors. Biomaterials (2011) 32: 144-51.
(20) Behnam B, Shier WT, Nia AH, Abnous K and
Ramezani M. Non-covalent functionalization of
single-walled carbon nanotubes with modified
polyethyleneimines for efficient gene delivery. Int. 
Karimi A et al. / IJPR (2019), 18 (Special Issue): 221-236
J. Pharm. (2013) 454: 204-15.
(21) Robinson JT, Welsher K, Tabakman SM, Sherlock
SP, Wang H, Luong R and Dai H. High performance
in-vivo near-IR (> 1 μm) imaging and photothermal
cancer therapy with carbon nanotubes. Nano Res.
(2010) 3: 779-93.
(22) Bates K and Kostarelos K. Carbon nanotubes
as vectors for gene therapy: past achievements,
present challenges and future goals. Advanced
drug delivery reviews. (2013) 65: 2023-33.
(23) Nepal D and Geckeler KE. Proteins and carbon
nanotubes: close encounter in water. Small. (2007)
3: 1259-65.
(24) Huang Z, Xi L, Subhani Q, Yan W, Guo W and
Zhu Y. Covalent functionalization of multi-walled
carbon nanotubes with quaternary ammonium
groups and its application in ion chromatography.
Carbon. (2013) 62: 127-34.
(25) Nunes A, Amsharov N, Guo C, Van den Bossche J,
Santhosh P, Karachalios TK, Nitodas SF, Burghard
M, Kostarelos K and Al-Jamal KT. Hybrid
Polymer-Grafted Multiwalled Carbon Nanotubes
for In-vitro Gene Delivery. Small. (2010) 6: 2281-
(26) Fisher JW, Sarkar S, Buchanan CF, Szot CS,
Whitney J, Hatcher HC, Torti SV, Rylander CG and
Rylander MN. Photothermal response of human
and murine cancer cells to multiwalled carbon
nanotubes after laser irradiation. Cancer Res.
(2010) 70: 9855-64.
(27) Chou H-T, Wang T-P, Lee C-Y, Tai N-H and Chang
H-Y. Photothermal effects of multi-walled carbon
nanotubes on the viability of BT-474 cancer cells.
Mater. Sci. Eng. C. (2013) 33: 989-95.
(28) Graham EG, MacNeill CM and Levi-Polyachenko
NH. Quantifying folic acid-functionalized multiwalled carbon nanotubes bound to colorectal
cancer cells for improved photothermal ablation. J.
Nanoparticle Res. (2013) 15: 1-12.
(29) Zhou F, Xing D, Ou Z, Wu B, Resasco DE and
Chen WR. Cancer photothermal therapy in the
near-infrared region by using single-walled carbon
nanotubes. J. Biomed. Opt. (2009) 14: 021009--7.
(30) Jeyamohan P, Hasumura T, Nagaoka Y, Yoshida
Y, Maekawa T and Kumar DS. Accelerated killing
of cancer cells using a multifunctional singlewalled carbon nanotube-based system for targeted
drug delivery in combination with photothermal
therapy. Int. J. Nanomed. (2013) 8: 2653.
(31) Burke A, Ding X, Singh R, Kraft RA, LeviPolyachenko N, Rylander MN, Szot C, Buchanan
C, Whitney J and Fisher J. Long-term survival
following a single treatment of kidney tumors with
multiwalled carbon nanotubes and near-infrared
radiation. Proc. Natl. Acad. Sci. (2009) 106:
(32) Torti SV, Byrne F, Whelan O, Levi N, Ucer B,
Schmid M, Torti FM, Akman S, Liu J and Ajayan
PM. Thermal ablation therapeutics based on CNx
multi-walled nanotubes. Int. J. Nanomed. (2007) 2: