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 Artificial Cells, Nanomedicine, and Biotechnology
An International Journal

ISSN: 2169-1401 (Print) 2169-141X (Online) Journal homepage: http://www.tandfonline.com/loi/ianb20

Development and evaluation of targeting ligand-

anchored CNTs as prospective targeted drug
delivery system

Sumandeep Kaur, Neelesh Kumar Mehra, Keerti Jain & Narendra Kumar Jain

To cite this article: Sumandeep Kaur, Neelesh Kumar Mehra, Keerti Jain & Narendra Kumar
Jain (2016): Development and evaluation of targeting ligand-anchored CNTs as prospective
targeted drug delivery system, Artificial Cells, Nanomedicine, and Biotechnology

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 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY, 2016
http://dx.doi.org/10.3109/21691401.2016.1146728

RESEARCH ARTICLE

Development and evaluation of targeting ligand-anchored CNTs as prospective

targeted drug delivery system
Sumandeep Kaura, Neelesh Kumar Mehraa,b, Keerti Jaina and Narendra Kumar Jaina
a
Pharmaceutical Nanotechnology Research Laboratory, ISF College of Pharmacy, Moga, Punjab, India; bDepartment of Pharmaceutical Sciences,
Pharmaceutics Research laboratory, R. H. S. Gour University, Sagar, Madhya Pradesh, India

ABSTRACT ARTICLE HISTORY

Our main investigation in the present research was to developt and evaluate targeting ligand- Received 24 September 2015
anchored multiwalled carbon nanotubes (MWCNTs) as prospective targeted drug delivery system, Revised 21 January 2016
with a special focus on the MWCNTs surface functionalization (FA-PEG bis-amine functionalized, Accepted 22 January 2016
Downloaded by [Texas A&M University Libraries] at 09:42 18 February 2016

carboxylated MWCNTs). In vitro release of 5-fluorouracil (5-FU) was studied at pH 7.4 phosphate Published online
buffer and 5.5 acetate buffer, which displayed initial faster followed by sustained release up to 16 February 2016
900 min. Further, 5-FU/FA-PEG bis amine-MWCNTs was found to be long circulating, prolonged KEYWORDS
half-life and increased drug accumulation in target tissue. 5-Fluorouracil; carbon nano-
tubes; folic acid; ligands;
PEGylation; targeting; tumor

Introduction (Mehra and Jain 2015a,b, 2016, Mehra et al. 2015). As literature
suggests that the multifunctional CNTs have higher surface
Nanotechnology involves research and technology develop-
area and available major and minor grooves for loading and
ment at atomic, molecular, or macromolecular levels and the
conjugation of high drug loading and entrapment capability as
use of functionalized nanostructures, nanodevices, and nano-
compared with other existing nanomaterials.
systems that take advantage of specific properties of matter,
Pristine CNTs usually contain metallic particles, amorphous
which exist at the nanoscale. It plays significant role in
carbon, and multishell impurities in large amounts. Purification
medicine, especially in the management of severe diseases
must be conducted before the utilization of nanotubes to
like cancer, leishmaniasis, AIDS, and diabetes (Agarwal et al.
remove and separate impurities from nanotubes for drug
2009). Nanotechnology can be exploited to deliver drugs,
delivery and targeting (Huczko 2002, Iijima and Ichihashi 1993,
peptides, proteins, and nucleic acids using various delivery
Ren et al. 2012). Different methods used for the purification of
systems and plays a significant role in drug delivery and
CNTs are cutting (cutting of CNTs into smaller pieces) (Jain et al.
targeting of pharmaceutical, therapeutic, and diagnostic agents
2009), microwave treatment (controlled temperature and
(Allard-Vannier et al. 2012, Bethune et al. 1993).
pressure conditions with a minimal need for solvents) (Jain
Carbon nanotubes (CNTs) were fully described by Sumio
et al. 2009), vacuum oven (Kayat et al. 2016), ultrasonication
Iijima (Iijima 1991) of Nippon Electronic Company in 1991 and it
(separate the impurities from pristine CNTs) (Li et al. 2007),
represents unique and outstanding physicochemical properties
chemical oxidation (conjugation of CNTs with biomolecules by
in drug delivery and targeting (Hirlekar et al. 2009, Mehra et al.
using HNO3, H2O2:NH4OH, HNO3:H2SO4, and HNO3: HCl) (Lodhi
2008, Mehra and Palakurthi 2015, Pruthi et al. 2012). CNTs
et al. 2013, Mehra and Jain 2013, Spinato et al. 2016).
mediated targeted and controlled drug delivery has aroused
In the present research study, our main aim was to the
growing awareness as precious, promising nanoarchitecture
development and evaluation of ligand-anchored MWCNTs for
due to its unique physicochemical properties, including a high
targeting to breast cancer with a spatial focus on the MWCNTs
aspect ratio, ultralight weight, high biocompatibility, and huge
surface functionalization (FA-PEG bis amine functionalized and
surface area in the treatment of cancer (Bhadra et al. 2003, Cao
carboxylated MWCNTs) and compared it with free 5-FU
and Feng 2008, Chiang et al. 2001, Das et al. 2013, Datsyuk et al.
solution. 5-FU-loaded purified carboxylated MWCNTs and FA-
2008, De la Cruz et al. 2012).
PEG bis amine-MWCNTs was developed and evaluated the in
CNTs are insoluble in water (hydrophobic in nature) or
vitro release, ex vivo and in vivo study.
organic solvents, nonbiodegradable, have short blood circula-
tion half-life (33.5 h) and are excreted through urine (94%) and
feces (6%) (Huang et al. 2011, Sharma et al. 2015). Depending Materials and methods
upon their diameter, lengths, and the presence of number of Raw (Pristine) MWCNTs were procured from Sigma Aldrich Pvt.
walls, CNTs can be single-, double-, triple-, multiwalled CNTs Ltd. 5-FU was received as a benevolent gift from United Biotech,

CONTACT Neelesh Kumar Mehra neelesh81mph@gmail.com Department of Pharmaceutical Sciences, Pharmaceutics Research laboratory, R. H. S. Gour
University, Sagar, Madhya Pradesh, India

2016 Taylor & Francis

 2 S. KAUR ET AL.
New Delhi, India. PEG-bis amine was purchased from Sigma
Aldrich Pvt. Ltd. Dulbeccos modified Eagle medium (DMEM) was
obtained from Hi-Media, Mumbai. Dicyclo hexyl carbodiimide
(DCC), dimethyl sulfoxide (DMSO), N-hydroxy succinimide (NHS),
nitric acid, ammonium hydroxide, hydrogen peroxide, and folic
acid (FA) were purchased from Lobachemie Pvt. Ltd. Ultrapure
water was purchased by Millipore System and other reagents
were obtained from the Lobachemie and used as received.
Purification of procured MWCNTs
The procured MWCNTs were purified by selective oxidation
method as reported by Li et al. (2007) with slight modifications.
Initially, pristine MWCNTs were purified using vacuum oven
(kept inside the Hot Air Sterilizer, New Delhi, India at 250 2  C
for 1 h) (Jain et al. 2009), then microwave (kept in the domestic
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microwave at 100 2  C for 15 min) (Mehra et al. 2014) and
subsequently strong acid treatment (H2SO4:HNO3 1:3) followed
by NH4OH and H2O2 to remove any metallic or amorphous
impurities from the unpurified carboxylated MWCNTs (Mittal
et al. 2010, Yudianti et al. 2011). The particle size and
polydispersity index (PDI) of purified MWCNTs were character-
ized by zeta sizer (Delsa Nano C particle Analyzer, Beckman
coulter). Purified MWCNTs was confirmed by FTIR spectroscopy
(Parkin Elmer, Shelton, CT).
Dispersion of MWCNTs in surfactants
The MWCNTs (0.50 mg) were dispersed in 0.5 mL of different
surfactants (sodium lauryl sulfate, Tween-80, and Triton X-100)
and 2 mL of distilled water and sonicated (Steryl Medi Equip,
40,050, India) for 2 h. The different dispersions were visualized
at different time intervals (Mehra et al. 2014).
Conjugation of FA-PEG bis amine to carboxylated
MWCNTs
Activation of folic acid
Folic acid (FA) (1 g) was dissolved in DMSO:TEA (40:0.5 mL) was
added in a reaction vessel and mixture of N-hydroxy
succinimide (NHS) (0.52 g) and N,N-dicyclo hexyl carbodiimide
(DCC) (0.50 g) was added with continuous stirring (at room
temperature in dark) for 18 h (Mehra and Jain 2013). For the
removal of precipitated by product (dicyclohexylurea; DCU)
mixture was filtered. Further, triethylamine (TEA) was removed
by evaporation under reduced pressure (temperature 20  C;
pressure 400 Pascal; rpm-50), and remaining solution
(activated FA; FA-NHS) was stored at 20  C. The obtained
product was characterized through FT-IR spectroscopy.
Conjugation of activated FA and PEG bis amine
The activated FA (FA-NHS) was conjugated to PEG-bis amine
(Sigma Aldrich, St. Louis, MO) according to previously reported
method after slight modifications (Mehra and Jain 2013). The
activated ester FA-NHS (8 mg) was mixed with 112.5 mg of PEG
bis amine (Sigma Aldrich), in the presence of DMSO (40 mL) and
TEA (4.0 mL) in the reaction vessel, after which the mixture was
magnetically stirred continuously at 100 rpm for 24 h. Folate-
conjugated PEG bis amine (FA-PEG bis amine) as pale yellow
solution was detected using UV-Vis spectrophotometer (lmax
363 nm) (Mehra et al. 2015, Mehra and Jain 2013).
Preparation of FA-PEG bis amine/MWCNTs
Carboxylated MWCNTs (10 mg) were dispersed in the mixture of
EDC and DMSO (6.41 mg/mL) in the reaction bottle and FA-PEG
bis amine (3 mL) was added to it with continuous magnetic
stirring for 6 h at 100 rpm, after which the mixture was stirred for
5 days. The remaining product was purified by dialysis (56 kDa,
HiMedia, Mumbai, India) to remove the un-conjugated FA-PEG
bis amine. The purified product (FA-PEG bis amine/MWCNTs)
was collected and characterized by FTIR spectroscopy (Mehra
et al. 2015, Mehra and Jain 2013, Zheng et al. 2009).
Drug loading
To the best of our knowledge, 5-FU was used for the first time in
functionalized MWCNTs. 5-FU (5 mg) was added to the mixture
of acetone (0.2 mL) and TEA (0.3 mL). The solution was magnet-
ically stirred vigorously for 23 min and the dispersion of
MWCNTs (5 mg) in phosphate buffer pH 7.4 (2.5 mL) was
added in the drug solution with continuous stirring for overnight
(24 h). The amount of bound drug was estimated spectro-
photometerically ( Bhadra et al. 2003, Huang et al. 2011, Mehra
and Jain 2013, Tavakolifard et al. 2015). For loading of 5-FU in FA-
PEG bis amine/MWCNTs conjugate 5-FU (25 mg) was added in
the dispersion of FA-PEG bis amine/MWCNTs (5 mL). The 5-FU-
FA-PEG bis amine/MWCNTs mixture was magnetically stirred
over night for 24 h. This conjugate was dialyzed under strict sink
conditions at 10 min (to remove the free drug from formulation),
which was then estimated spectrophotometerically (Shimadzu,
Japan) to determine the amount of drug bound with system.
In vitro study
In vitro release of 5-FU from 5-FU/MWCNTs and 5-FU/FA-PEG
bis amine-MWCNTs formulation was studied in phosphate
buffer (pH 7.4) and acetate buffer (pH 5.0) at room temperature
(37 0.5  C) in 5, 10, 15, 20, 30, 60, 90, 120, 150, 180, and
900 min time points. 5-FU-loaded formulations (5-FU/MWCNTs
and 5-FU/FA-PEG bis amine-MWCNTs) was filled in dialysis
membrane (MWCO 56 kDa, Hi Media, India) separately,
hermetically tied at both ends and instantly located into the
receptor media maintaining strict sink conditions with steady
stirring using magnetic stirrer at room temperature (100 RPM;
Remi, Mumbai, India). The samples were collected at different
time points and concentration of 5-FU was determined in
triplicate by UV/Visible spectrophotometer at lmax 266 nm (UV/
Vis, Shimadzu 1601, Kyoto, Japan) (Ren et al. 2012, Shi et al.
2009, Singh et al. 2008).
Cell line study
Cytotoxicity studies
MCF-7 (Michigan Cancer Foundation-7) human breast cancer
cell was procured from National Center of Cell Sciences (NCCS),

Pune, India, which is of folate over expression. Cells were
cultured in DMEM containing 10% FBS and 1%
penicillin/streptomycin. Cells were cultured in humidified
environment at 37  C with CO2. MCF-7 cells were incubated in
96-well plates for 12 h, the old medium was removed, and after
incubation, cells were incubated in media containing 5-FU-
loaded MWCNTs and FA-PEG bis amine/MWCNTs at the
equivalent drug concentration (110 mg/mL). MTT assay was
used to measure the cell viability at given time intervals. The
absorbance of each well was measured by microplate reader
(ELISA) with wavelength at 266 nm (Yoong et al. 2014).
Cell uptake study
Folate receptors overexpressed MCF-7 cells were cultured on
glass cover slips placed into tissue culture plates and incubate
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the cell overnight. After overnight growth, supernatants from
the culture of 5-FU-loaded MWCNTs and FA-PEG bis
amine/MWCNTs at 37  C were added. Upon incubation for
2 h, cells were washed and observed under fluorescence
microscopy (FSM) after excitation at 266 nm.
In vivo study
The Wistar rats of either sex (200250 g) were used for present
animal studies in accordance with the guidelines by Committee
for the Prevention, Control and Supervision of Experimental
animals (CPCSEA) ISFCP/IAEC/CPCSEA/Meeting No 15/2015/
Protocol No.261 from ISF College of Pharmacy, Moga Punjab,
India. All the experimental protocols were approved by the
Institutional Animal Ethics Committee.
Analysis of pharmacokinetic profile after dosing
The pharmacokinetic parameters were calculated after i.v.
administration of free 5-FU and 5-FU/MWCNTs and 5-FU-FA-
PEG bis amine/MWCNTs nanoformulations with the calculated
i.v. dose (3.0 mg/kg body weight dose). The blood samples
were collected from the retro-orbital plexus of eyes into the Hi-
anti-clot blood collecting vials at predetermined time points
up to 24 h. Supernatant was collected after centrifugation,
Figure 1. FTIR spectrum of oven-treated MWCNTs (at 250  C for 30 min).
ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 3
trichloroacetic acid (TCA) in methanol (10% w/v) was added,
vortexed, and ultracentrifuged (Rami, New Delhi). The clear
supernatant was collected after centrifugation and 5-FU
concentration was determined by high-performance liquid
chromatography (HPLC) method and different pharmacokinetic
parameters were calculated by Kinetica, Version 5.0 (Mattos et
al. 2013, Mehra et al. 2014, Singh et al. 2008).
Biodistribution study
The in vivo biodistribution of the 5-FU/MWCNTs, 5-FU-FA-PEG
bis amine/MWCNTs formulations and free 5-FU were studied on
wistar rats. The free 5-FU, 5-FU/MWCNTs, and 5-FU-FA-PEG bis
amine/MWCNTs conjugates were administered i.v. through tail
vein route according to body weight of animals. Rats were
carefully sacrificed after predetermined time intervals 6, 12, and
24 h for the collection of organs such as liver, lung, kidney, and
spleen immediately. Organs were homogenized after addition
of chloroform (CHCl3) and methanol (CH3OH) mixture and
ultracentrifuged at 2000 rpm for 25 min. After centrifugation,
collect the solid-dried residue. Concentration of 5-FU were
determined by HPLC (Shimadzu, C18, Japan) method, wherein
mobile phase consisted of water/acetonitrile (90:10; v/v) (Singh
et al. 2008).
Statistical analysis
Data were expressed as the means with 95% confidence
intervals. Statistical test were performed with one-way analysis
of variance (ANOVA) test. For all tests, P values less than 0.05
(P50.05) were considered to be statistically significant.
Results and discussion
Pristine CNTs are not suitable due to intrinsic aqueous
insolubility and existence of impurities. The procured pristine
MWCNTs from Sigma Aldrich Pvt. Ltd. were purified in an oven,
microwave and afterward strong acid treatment (H2SO4:HNO3)
(separate the impurities metallic or amorphous) followed by
NH4OH and H2O2 and cut (shorten nanotubes) prior to use.
Figures 1 and 2 show the FTIR of oven which shows the peaks
 4 S. KAUR ET AL.
Figure 2. FTIR spectrum of acid (H2SO4:HNO3::NH4OH:H2O2)-treated MWCNTs.
Downloaded by [Texas A&M University Libraries] at 09:42 18 February 2016
Table 1. Effects of sonication on particle size and polydispersity index on
functionalization of CNTs.
Treatment Particle size (nm) PDI
Oven treatment 881.10 5.52 0.436 0.02
Microwave treatment 863.42 8.45 0.381 0.12
Acid treated (H2SO4:HNO3::NH4OH:H2O2) 706.12 2.42 0.303 0.04
Acid treated (H2SO4:HNO3) reflux 8 h 570.94 6.54 0.254 0.14
of OH and NH stretching at 3447.12 and 2945.58 cm1,
respectively, and acid-treated MWCNTs depicts the peaks of
COOH (3062.11 cm1), asymmetric stretching of C O
(1633.58 cm1) and CO stretch (1289.12 cm1) and Table 1
shows the particle size of purified CNTs. The longer nanotubes
are unable to enter most of the cancerous cells and may be
toxic. Oxidation is one of the most common techniques for
hydrophilic functionalization of the nanotubes through strong
oxidizing acids.
The dispersion characteristics of MWCNTs (0.50 mg) in differ-
ent surfactants (sodium lauryl sulfate, Tween-80, and Triton
X-100) are shown in Figure 3. The surface of the MWCNTs was
modified using different surfactants, in presence of sonication.
Sodium lauryl sulfate (SLS), showed the maximum dispersion of
MWCNTs, CNTs were sediment in Tween-80. The dispersion of
MWCNTs in different surfactants after 48 h is as follows:
Sodium lauryl sulfate > Triton X  100 > Tween 80
The FTIR spectrum of procured FA is shown in Figure 4. FTIR
spectrum of the activated folic acid shows the peaks at 1651,
1712, and 1557 cm1 which shows the presence of CH
stretching, NH stretching, and asymmetric stretching of C O,
respectively. Folic acid and PEG bis amine conjugate was
prepared from PEG bis amine by successive conjugation with
activate folic acid. The FA-PEG bis amine conjugate was
obtained as a pale yellow liquid. FTIR spectrum of conjugate
demonstrates that folic acid was indeed covalently linked to
the PEG via amide bond (Figure 5). FTIR depicts the peaks of
amide I C O (1651 cm1), amide II (1i bond) (1644.63 cm1),
amide III (2i bond). FTIR spectrum of FA-PEG bis amine/MWCNT
shows the peaks of aromatic compounds indicated the
presence of FA and CO stretch of ether linkage shown in
Figure 6.
The drug-loading efficiency is the significant, precondition
characteristic in development and characterization of targeted
drug delivery system. The drug entrapment efficiency was
studied following a modified dialysis diffusion technique and
was found to be significantly higher. The entrapment of 5-FU in
MWCNTs was monitored by UV/Visible absorption to confirm
indirectly, the drug entrapment in MWCNTs and FA-PEG bis
amine, during which the characteristic absorption peak of 5-FU
at 266.61 nm was shifted, indicative of interaction between
5-FU and MWCNTs. The entrapment efficiency was found to be
99.8% in MWCNTs and 99.5% in FA-PEG bis-amine MWCNTs and
5-FU-MWCNTs.
The in vitro behavior of 5-FU/MWCNTs and 5-FU-FA-PEG bis
amine/MWCNTs in phosphate buffer (pH 7.4) and acetate buffer
(pH 5.0) using dialysis bag method is shown in Figure 7, which
displayed an initial faster followed by sustained pattern. In the
acidic pH-sustained release pattern due to the ionization of
5-FU, our release profile was in good agreement with
previously published reports (Bhadra et al. 2003, Mehra and
Jain 2013, Mehra et al. 2014, Ren et al. 2012,Yoong et al. 2014).
The methylthiazole tetrazolium (MTT) cytotoxicity assay was
performed by the cleavage of tetrazolium salt [{3(4,5 dimethyl
thiazole-2 yl)-2,5-diphenyl tetrazolium bromide} (MTT)] to a
blue formazan derivative by living cells. The MTT assay was
performed to determine the cancer-targeting propensity of the
5-FU laden developed optimized nanotubes formulation using
MCF-7 (human breast cancer) cell line and compared with FA-
PEG bis amine-MWCNTs and free 5-FU solution. The cytotoxicity
assay clearly demonstrated that upon increasing the concen-
tration from 0.01 to 10 mg/mL. The percent viability of the
cancerous cells was decreased owing to apoptosis by
intercalating 5-FU (Figure 8). Our cytotoxicity results are in
accordance with the previous published reports.
The qualitative cellular uptake of 5-FU and 5-FU-loaded
MWCNTs formulations is studied and presented in Figure 9. The
5-FU-FA-PEG bis amine/MWCNTs shows higher fluorescence
intensity as compared with other formulation and control
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 5

Figure 3. Dispersion of multiwalled carbon nanotubes in different surfactants.

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Figure 4. IR spectrum of procured folic acid.

Figure 5. FTIR spectrum of FA-PEG bis amine.

 6 S. KAUR ET AL.
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Figure 6. FTIR spectrum of FA-PEG bis amine/MWCNTs.

Figure 7. % Cumulative release from: (A) 5-FU-MWCNTs (B) 5-FU-FA-PEG-MWCNTs formulations in PBS 7.4 and acetate buffer 5.0.

100
90 fluorescence intensity clearly suggests the higher uptake of
80 the 5-FU/FA-PEG bis amine-MWCNTs formulations, possibly due
% Cell viabiility

70
60
to the receptor-mediated endocytosis (RME) as well nanonee-
50 dle-specific (passive diffusion) mechanism.
40 In vivo bioavailability and pharmacokinetic studies of free
30 free 5-FU
20 5-FU/MWCNTs
5-FU, 5-FU-MWCNTs, and 5-FU-FA-PEG bis amine-MWCNTs were
10 5-FU/FA-PEG-MWCNTs conducted on Wistar rats. Following i.v. dosage of sample,
0 blood samples were collected at specified time points and 5-FU
0 0.01 0.1 1 10
concentration was determined in plasma by HPLC method.
Concentration (g/mL)
Results obtained are plotted with plasma concentration versus
Figure 8. Percent cell viability of MCF-7 cell after treatment with free 5-FU, 5-FU/ time as shown in Figure 10. Pharmacokinetic parameters were
MWCNTs and 5-FU/MWCNTs (n 3). determined using noncompartment modeling as summarized
in Table 2. The elimination half-life (t1/2) of 5-FU-FA-PEG bis
group. Similarly, higher cell uptake fluorescence intensity was amine/MWCNTs, 5-FU/MWCNTs, and free 5-FU was found to be
observed for 5-FU/FA-PEG bis amine-MWCNTs, as compared 16.4754, 8.6439, and 5.6192, while MRT was found to be
with 5-FU/MWCNTs and free 5-FU. The observed higher 17.0036, 10.3746, and 5.2347, respectively. Thus, indicating an
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 7

Figure 9. Qualitative cell uptake of the free 5-FU, 5-FU/MWCNTs and 5-FU-FA-PEG-MWCNTs in MCF-7 cells.
Downloaded by [Texas A&M University Libraries] at 09:42 18 February 2016

kidney (Free-5-FU) kidney (5-FU-CNTs) kidney (5-FU-FA-PEG-CNTs)

lung (Free-5-FU) lung (5-FU-CNTs) lung (5-FU-FA-PEG-CNTs)
spleen (Free-5-FU) spleen (5-FU-CNTs) spleen (5-FU-FA-PEG-CNTs)
liver (Free-5-FU) liver (5-FU-CNTs) liver (5-FU-FA-PEG-CNTs)
40
Concentration (g/g) organ

liver
30 kidney

20
lung
spleen
10

0
6 12 24 6 12 24 6 12 24 6 12 24
time (hr)

Figure 10. Biodistribution patterns of free 5-FU, 5-FU-MWCNTs and 5-FU-FA-PEG bis amine-MWCNTs in different organs.

tissue distribution of free 5-FU, 5-FU-MWCNTs, and 5-FU-FA-

Table 2. Pharmacokinetic parameters of free 5-FU, 5-FU-MWCNTs, and 5-FU-FA- PEG bis amine-MWCNTs after i.v. administration in rats. Tissue
PEG bis amine-MWCNTs.
biodistribution study was conducted to evaluate the drug
Formulations code
delivery at the different sites of interest like liver, spleen,
5-FU-FA-PEG bis
Pharmacokinetic parameters Free 5-FU 5-FU-MWCNTs amine-MWCNTs kidneys, and lungs. The free 5-FU was primarily accumulated
Cmax (mg/mL) 0.3644 0.4810 0.5069 progressively in liver up to 6-h postinjection. After 12 h, only
Tmax (hr) 1.0000 1.0000 1.0000 0.51 0.09 mg/g of drug concentration was found in liver,
AUCtotal 1.4725 2.6036 3.7362
whereas spleen, kidney, and lungs were found to have drug
% AUC 0.2292 1.4941 9.8714
HVD 1.9275 1.9175 2.3665 levels up to 0.25 0.03, 0.39 0.01, and 0.51 0.02 mg/g,
T1/2 (h) 5.6192 8.6439 16.4754 respectively. When 5-FU-MWCNTs and 5-FU-FA-PEG bis
MRT (h) 5.2327 10.3746 17.0036s
amine-MWCNTs was administered, appreciably lesser amounts
were present in spleen and lungs. The purified MWCNTs that
are devoid of free terminal functional groups, readily goes into
the liver. According to previously published report, after i.v.
overall increase in bioavailability of 5-FU due to the targeted injection, CNTs are mainly excreted by urine and feces. The 5-
delivery through ligands conjugated MWCNTs. FU-MWCNTs and 5-FU-FA-PEG bis amine-MWCNTs is mainly
A comparative biodistribution study was performed among accumulated in liver. The appreciable amount of drug from the
the free 5-FU, 5-FU-MWCNTs, and 5-FU-FA-PEG bis amine- developed CNTs formulation was found in liver. No significant
MWCNTs formulations administered i.v. to compare the amount accumulation of 5-FU-MWCNTs and 5-FU-FA-PEG bis amine-
of 5-FU that reaches different tissue like liver, spleen, kidney, MWCNTs was observed in spleen and lungs. As the folate
and lungs at different time intervals. Figure 10 represents the conjugation was carried out on the carboxylated MWCNTs, the
 8 S. KAUR ET AL.
dispensability and hydrophilicity of MWCNTs were enhanced
significantly as revealed by dispersion and loading experimen-
tation. In general, 5-FU-MWCNTs and 5-FU-FA-PEG bis amine-
MWCNTs formulation was administered by i.v. route; the
concentration of drug was comparatively lesser in all the
tissues than the free 5-FU in the initial 6 h, slight increase in 12 h
and reduction due to the clearance in 12-h postinjection. This
outcome could be attributed to the fact that 5-FU-MWCNTs
and 5-FU-FA-PEG bis amine-WWCNT released appreciable drug
in the systemic circulation than in any other tissue. The
MWCNTs are nonbiodegradable in nature and known to be
excreted by biliary pathway from the liver through the bile
duct, intestine, which ends up in feces.
Conclusion
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The multiwalled carbon nanotubes (MWCNTs) have been
continuously attracting increasing consideration in targeted
and controlled drug delivery. As we know that the functiona-
lized CNTs provide greater stability as well as loading efficiency
and better and alternative platform in biomedical applications
including targeted drug delivery. In the present investigation,
our main aim was to develop, evaluate, and investigate the
cancer-targeting propensity of the 5-FU-loaded functionalized
MWCNTs (PEGylation) using human breast cancer cells. The
in vitro, ex vivo and in vivo studies clearly revealed that the
5-FU/PEG-MWCNTs showed promising potential in cancer
targeting and effectively kill cancerous cells. The pharmacokin-
etics studies also showed that the developed MWCNTs formu-
lations are long circulating (stealth) in nature and increases
dispersibility of CNTs. Finally, it can conclude that the 5-FU-
loaded FA-PEG bis amine/MWCNTs formulation showed effi-
cient 5-FU release at desired site.
Disclosure statement
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
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