Natural deep eutectic solvent supported targeted solid-liquid polymer carrier for breast cancer therapy

Article abstract of DOI:10.1039/d0ra03790g

Solid-liquid nanocarriers (SLNs) are at the front of the rapidly emerging field of medicinal applications with a potential role in the delivery of bioactive agents. Here, we report a new SLN of natural deep eutectic solvent (NADES) and biotin-conjugated lysine-polyethylene glycol copolymer. The SLN system was analyzed for its functional groups, thermal stability, crystalline nature, particle size, and surface morphology through the instrumental analysis of FT-IR, TGA, XRD, DLS, SEM, and TEM. Encapsulation of PTX (paclitaxel) and 7-HC (7-hydroxycoumarin) with the SLN was carried out by dialysis, and UV-visible spectra evidenced the drug loading capacity and higher encapsulation efficiency obtained. The enhanced anticancer potential of PTX- and 7-HC-loaded SLN was assessedin vitro, and the system reduces the cell viability of MDA-MB-231 cells. The PTX- and 7-HC-loaded SLN system was investigated in a breast cancer-induced rat modelvia in vivostudies. It shows decreased lysosomal enzymes and increased levels of caspase to cure breast tumors. It very well may be reasoned that the designed PTX- and 7-HC-loaded SLN system has strong anticancer properties and exhibits potential for delivery of drug molecules in cancer treatment.

Full text of DOI:10.1039/d0ra03790g

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PAPER
Natural deep eutectic solvent supported targeted
solid–liquid polymer carrier for breast cancer
therapy†
Cite this: RSC Adv., 2020, 10, 36989
a
b
Xianfu Sun, Periyakaruppan Pradeepkumar, Naresh Kumar Rajendran,c
*
Harshavardhan Shakila,d Nicolette Nadene Houreld,c Dunia A. Al Farraj,e
Yousif M. Elnahas,f Nandhakumar Elumalaig and Mariappan Rajan
*
b
Solid–liquid nanocarriers (SLNs) are at the front of the rapidly emerging field of medicinal applications with
a potential role in the delivery of bioactive agents. Here, we report a new SLN of natural deep eutectic
solvent (NADES) and biotin-conjugated lysine–polyethylene glycol copolymer. The SLN system was
analyzed for its functional groups, thermal stability, crystalline nature, particle size, and surface
morphology through the instrumental analysis of FT-IR, TGA, XRD, DLS, SEM, and TEM. Encapsulation of
PTX (paclitaxel) and 7-HC (7-hydroxycoumarin) with the SLN was carried out by dialysis, and UV-visible
spectra evidenced the drug loading capacity and higher encapsulation efficiency obtained. The
enhanced anticancer potential of PTX- and 7-HC-loaded SLN was assessed in vitro, and the system
reduces the cell viability of MDA-MB-231 cells. The PTX- and 7-HC-loaded SLN system was investigated
in a breast cancer-induced rat model via in vivo studies. It shows decreased lysosomal enzymes and
increased levels of caspase to cure breast tumors. It very well may be reasoned that the designed PTX-
and 7-HC-loaded SLN system has strong anticancer properties and exhibits potential for delivery of drug
molecules in cancer treatment.
Received 27th April 2020
Accepted 11th August 2020
DOI: 10.1039/d0ra03790g
rsc.li/rsc-advances
reduce some disadvantages over conventional anticancer
treatments such as poor solubility, low biodegradability, toxic
Introduction
nature, and poor intracellular penetration, retention time and
sustainable release of encapsulated drugs.9
signicant
Recent research development in medicinal applications has
advanced through the improvement of new methods and
materials that are more biodegradable, biocompatible, and less
toxic.1,2 There is an enormous attempt to explore the role/
function of synthetic biomaterials used in drug delivery appli-
cations.3 Currently, synthetic and natural polymers, metal
complexes, peptides, proteins, and hydrogels are used for the
development of anticancer drug delivery systems,4–8 which
A
number of nanoparticles such as inorganic nanoparticles or
other polymeric/liquid nanoparticles have also been used in
developing drug carrier systems. The drug carrier leads to the
fabrication of a nanocarrier, and most of these are made up of
synthetic polymers and not liquid-based carriers.10
Researchers have paid more attention to the advancement
of new carrier systems by using ILs (ionic liquids) and DESs
(deep eutectic solvents).11 Unfortunately, ILs and DESs have
exhibited thermal instability, low drug loading levels, low drug
release and solubility, and have a very weak interaction with
and a toxic nature in biological systems. This issue can be
overcome by the utilization of natural deep eutectic solvents
aDepartment of Breast, The Affiliated Tumor Hospital of Zhengzhou University,
Zhengzhou, Henan, 450008, China
bBiomaterials in Medicinal Chemistry Laboratory, Department of Natural Products
Chemistry, School of Chemistry, Madurai Kamaraj University, Madurai-625021,
Tamil Nadu, India. E-mail: rajanm153@gmail.com
cLaser Research Centre, Faculty of Health Sciences, University of Johannesburg, PO Box
(NADES).
A NADES is a highly biocompatible material
17011, Doornfontein 2028, South Africa
designed to serve as a carrier molecule that transports drugs to
a specic site without any side effects; it is a non-toxic solvent
prepared by secondary metabolites and does not affect the
drug release mechanism.12 Secondary metabolites like
phenolics, terpenoids, avonoids, and other natural
compounds are crucial for medicinal applications.13,14
Compared to organic solvents, the use of NADES has gained
attention in the synthesis of carrier systems.15–18
dDepartment of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj
University, Madurai-625021, India
eDepartment of Botany and Microbiology, College of Science, King Saud University,
Riyadh 11451, Saudi Arabia
fDepartment of Surgery, College of Medicine, King Saud University Medical City, King
Saud University, Riyadh, Saudi Arabia
gDepartment of Biochemistry, Sri Muthukumaran Medical College and Research
Institute, Chennai-600069, Tamil Nadu, India
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra03790g
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Citrus limon peels are rich in bioactive compounds, including
antioxidants, avonoids, vitamins, and prolinebetaine (PB). PB is
a natural bioactive compound, which has numerous biological
properties, including antifungal, antibacterial, and anticancer
activities.19,20 Anticancer activity is an essential property of PB and
is due to the presence of amine which is a highly reactive
compound that can readily react with biological compound
moieties such as hydroxyl groups. In this study, PB is isolated and
used for the synthesis of NADES in combination with lactic acid
(LA), which is an excellent electron donor/acceptor. The self-
assembled drug carrier system with PB is reported to target
tumor cells.21 However, there is the need for an extra efficient and
active targeting ligand to achieve better cellular uptake of drug-
loaded carriers. Different ligands such as polysaccharides, folic
acid, peptides, and biotin are involved in targeting drug carrier
synthesis.22 Among these, biotin is predominantly utilized as
a tumor-focusing group for numerous cancer chemotherapy
applications.23 Tumor cells need vitamins for their survival, and
speedily growing cancer cells overexpress the receptors for this
vitamin on their cell surface.
This paper reports for the rst time on the preparation of
NADES using secondary metabolites, and it is used as a biotin-
conjugated solid–liquid nanocarrier (SLN) for the encapsulation
of the anticancer drugs paclitaxel (PTX) and 7-hydroxycoumarin
(7-HC). The advantage of SLNs is the ability to incorporate
hydrophobic anticancer drugs in the inner core and hydrophilic
anticancer drugs on the outer core. The incorporation of PTX
and 7-HC in the SLN system will reduce side effects and improve
bioavailability. The anticancer activity of the synthesized PTX-
and 7-HC-loaded SLN system was investigated in vitro in a MDA-
MB-231 breast cancer cell line, and in vivo in a DMBA-induced
breast cancer rat model.
Isolation of prolinebetaine from Citrus limon peels
Citrus fruits (Citrus limon peels) were acquired from a nearby
market at Madurai, Tamil Nadu, India.24 The fruits were cleaned
with DD water and acetone. The fruit peels were chopped into
little pieces and dried at room temperature (27 ꢀC). Dried
samples were powdered into ne particles utilizing a blender.
Ground samples were then dissolved in various solvents such as
petroleum ether, chloroform, ethyl acetate, methanol, acetoni-
trile, and water for solubility optimization. Samples were loaded
onto a silica gel packed column (100–200 mesh). Pet ether was
used for initial elution. Fractions were removed for primary
compounds such as oils, fats, fatty acids, resin, and chlorophyll
compounds. Solvents were recovered through vacuum rotary
evaporation (Rotavapor® R-300).
Fractions were collected and used for further analysis.
Optimized and stabilized ethyl acetate and methanol in ratios of
70 : 30 and 50 : 50 were used for the collection of 4-hydrox-
yprolinebetaine and PB. The solvents from the collected
samples were evaporated at room temperature (27 ꢀC). The
derived compounds were identied by HPLC, 1H- and 13C-NMR,
and FT-IR analysis.
Synthesis of NADESs
NADESs were prepared according to a previously reported
method.25 Isolated and puried PB and LA were mixed in molar
ratios of 1 : 1, 1 : 2, 1 : 3, and 2 : 1 at 40 ꢀC. Mixtures were
magnetically stirred until a transparent homogenous liquid was
obtained. Aerward, the eutectic mixture was chilled to room
temperature. Ratios of 1 : 2 and 1 : 3 formed a clear solution,
while proportions of 1 : 1 and 2 : 1 did not create a clear solu-
tion, as is shown in ESI Fig. 1.† The formation of NADESs and
the chemical structures were identied through 1H and 13C
NMR and FT-IR spectroscopy, as shown in ESI Fig. 3 and 4.†
Experimental section
Materials
Synthesis of biotin-g-lysine
EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), LA
(lactic acid), NHS (N-hydroxysuccinimide), p-TsOH (p-toluene-
sulfonic acid), tin(II) 2-ethylhexanoate (Sn(Oct)2), and 7,12-
dimethylbenz[a] anthracene (DMBA) were procured from
Sigma-Aldrich, Mumbai, India. Lysine, polyethylene glycol-
6000, biotin, PTX, and 7-HC were purchased from Merck,
Mumbai, India. Petroleum ether (PE), dichloromethane (DCM),
ethyl acetate (EA), diethyl ether (DE), methanol, acetone,
acetonitrile (AN) and dimethyl sulfoxide (DMSO) were received
from Himedia Laboratories Pvt. Ltd, Mumbai, India (HPLC
grade). A human breast adenocarcinoma (MDA-MB-231) cell
line and normal mouse broblast cell line (L929) obtained from
the National Center for Cell Science, Pune, India were used for
this work. Caspase colorimetric assay kit was from R and D
Systems Inc., USA. The chemicals and solvents used were of
analytical grade and main clarity. In all the experiments,
double-distilled (DD) water was used as a solvent and washing
Biotin graed lysine was prepared by the condensation method
and carried out in the presence of EDC$HCl and NHS as per
previous literature.26 In general, biotin (0.2 g, 0.00136 mmol)
and lysine (0.33 g, 0.00136 mmol) were solubilized in DCM (15
mL). To this mixture, EDC$HCl (0.168 mL, 0.00136 mmol) and
NHS (0.15 g, 0.00136 mmol) were added drop-wise. The pH of
the reaction mixture was adjusted to pH 7.4 using 1 N NaOH
and stirred for 2 h at 45 ꢀC. Then, the reaction mixture was
precipitated using an organic solvent (acetone). The precipitate
was ltered using Whatman (0.2 mm) lter paper and dried in
a vacuum chamber. The obtained precipitate was dialyzed using
a dialysis membrane (MWCO-12000) in DD water for 22 h to
eliminate unreacted constituents.
Synthesis of biotin-g-lysine-co-polyethylene glycol (biotin-g-
lysine-co-PEG)
solution. The other chemicals were purchased from the Typically, biotin-g-lysine (1 g, 0.00268 mmol), p-TsOH (0.4 g,
different companies like BDH Division (Mumbai, India), Glaxo 0.00268 mmol), and polyethylene glycol-6000 (1 g) in 50 mL
Laboratories, Sisco Research Laboratories, Sarabhai Chemicals toluene were placed in a double neck round reaction ask with
(Vadodara, India), and SD Fine Chemicals (Mumbai, India).
a Dean–Stark apparatus.27 Then, Sn(Oct)2 (0.8 mL, 1.0857 mmol)
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was added drop-wise, and heated at 125 ꢀC for 24 h to reux the
mixture with continuous stirring and elimination of water. The
reaction mixture was cooled to room temperature (27 ꢀC);
subsequently, the reaction solution was dissolved in ether and
ltered to remove by-products. A Soxhlet apparatus was used for
polymer extraction for 24 h with an organic solvent (ethyl
acetate). Then, the obtained crude product was dried in
a vacuum and the chemical structure of biotin-g-lysine-co-PEG
was conrmed through FT-IR spectroscopy.
Encapsulation efficiency ð%Þ
total amount of drug ꢁ free amount of drug
¼
ꢃ 100
total amount of drug
(1)
Drug release study
The discharge of PTX and 7-HC from the nanocarrier was
transferred to the dialysis system and a physiological buffer
solution at pH values of 2.8, 5.5, and 7.4 was used at 27 ꢀC.
Briey, PTX- and 7-HC-loaded nanocarrier samples (30 mg) were
added to 5 mL of fresh PBS (pH 2.8, 5.5, and 7.4) in the dialysis
bag at room temperature. The dialysis membrane was incu-
bated in 50 mL PBS. PTX- and 7-HC-loaded SLN samples were
stirred using a magnetic stirrer at a constant rate (100 rpm), and
3 mL of the supernatant solution was extracted at standard time
intervals and supplemented with 3 mL of new buffer solution.
Drugs released into the medium were quantied by a UV-visible
spectrometer at lmax values of 270 nm and 320 nm (Shimadzu
1600, Japan).
NADES-based biotin-conjugated solid–liquid polymer in
solvent emulsion method
NADES-based biotin-conjugated polymeric SLN was prepared by
the method mentioned in our previous report.28 Initially, 1 g of
biotin-g-lysine-co-PEG was dissolved in 1 mL of NADES. This
reaction mixture was kept under continuous stirring for 1 h at
room temperature until the mixture became a homogeneous
solution and attained a viscous consistency. Nanocarriers were
formed using dialysis in aqueous solution, following a previous
report.29 Briey, 100 mg of NADES-biotin-g-lysine-co-PEG system
was dispersed in 1 mL DMSO solution and the solution was
placed into a dialysis bag and dialyzed against 250 mL of DD
water for 24 h to eliminate the DMSO solvent. The separated Swelling studies
SLN was used for further processing.
The swelling behavior of PTX- and 7-HC-loaded SLN (30 mg) was
analyzed in various buffer solutions at pH 2.8, 5.5, and 7.4 at
ꢀ
room temperature (27 C). At standard time intervals, samples
were removed and weighed. A stable weight was achieved and
Determination of self-assembly of carrier formation
The critical nanocarrier concentration (CMC) value of NADES- the following equation was used to determine the swelling
based biotin-conjugated solid–liquid polymer nanocarrier was behavior:
studied by uorescence spectroscopy through pyrene as a uo-
We ꢁ Wd
rescent moiety.30 In brief, various concentrations of NADES-
based biotin-conjugated solid–liquid polymer nanocarrier
(0.1 mL to 1.0 mL) were dissolved in 10 mL of DMSO solution.
Then, pyrene (0.1 mg) was solubilized in 5 mg mLꢁ1 methanol
and added to each test container of NADES-based biotin-
Swelling ð%Þ ¼
(2)
Wd
where We and Wd are the weights of swollen polymer and dried
nanocarriers, respectively.
conjugated solid–liquid polymer nanocarrier solution. The Biological characterizations
reaction solution was magnetically stirred at 27 ꢀC for 1 day, and
Cell culture. A human breast adenocarcinoma cell line
solvents vaporized. Aerwards, 5 mL of distilled water was
added individually in a sample tube. The uorescence of every
pyrene-containing NADES-based biotin-conjugated solid–liquid
polymer nanocarrier solution was determined by emission
spectra (spectrouorometer, Shimadzu F-4500, Japan) at
(MDA-MB-231) and a mouse broblast cell line (L929) were
purchased from the NCCS (National Center for Cell Science,
Pune, India). Cells were maintained in Dulbecco's modied
Eagle's medium supplemented with 10% fetal bovine serum,
and 1 unit per mL penicillin and 1 mg mLꢁ1 streptomycin, and
incubated at 37 ꢀC with 5% CO2 in a humidied chamber. Cells
ꢀ
a wavelength of 472 nm at 21 ꢂ 1 C.
were passaged every
3
to
4
days using trypsin/
ethylenediaminetetraacetic acid (EDTA) for detachment.
Encapsulation efficiency (EE)
Cell viability. MDA-MB-231 human breast adenocarcinoma
The NADES-based biotin-conjugated solid–liquid polymer and L929 normal mouse broblast cells were used to interrogate
nanocarrier (100 mg) was taken in a 25 mL beaker and PTX the cytotoxic effect of PTX- and 7-HC-loaded and unloaded SLN
(20 mg mLꢁ1) and 7-HC (20 mg mLꢁ1) dispersed in ethanol were system. Cells were seeded into 96-well plates at a seeding
added to the SLN and stirred for 1 h with a magnetic stirrer. For density of 1 ꢃ 105 cells per well and incubated for 24 h at 37 ꢀC
determining the drug encapsulation efficiency, the supernatant in 5% CO2. Various concentrations of PTX- and 7-HC-loaded
solution was collected at 10 min intervals and analyzed by UV- SLN system (10, 25, 50, 75, and 100 mg mLꢁ1) were added to
visible spectroscopy at lmax values of 270 nm and 320 nm wells and cells were cultured for 24 h. Aerwards, 25 mL of MTT
(Shimadzu UV 1800).16 EE was calculated with the following (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
equation:1
assay solution was added to each well and incubated for 5 h at
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37 ꢀC. The cell culture medium was measured at an absorption Government of India, and Institutional Animal Ethics
wavelength of 570 nm using a microplate reader (SpectraMax Committee (IEAC) Guidelines.
M3, Molecular Devices, USA). The corresponding cell viability
Gross observations and tumor volume. The change in the
(%) was correlated to normal cells. The following equation for body weight and tumor of animals was measured and recorded
cell viability was used for IC50 calculation:
OD of the treated cells
in grams. The following formula was used to calculate tumor
volume:
Cell viability ð%Þ ¼
OD of the untreated cells ðcontrolÞ
ꢃ 100
v ¼ 4/3pr12r2 (radius r1 < r2; r ¼ tumor diameter in mm/2)
(3)
Estimation of lysosomal marker enzymes. The enzymatic
activity of acid phosphatase and cathepsin-D was evaluated as
reported previously,31,32 and values were expressed as phenol/
protein min per mg and tyrosine/protein min per mg, respec-
tively. b-D-Glucuronidase was assayed by the method of Kawai
and Anno,33 and the activity was expressed as p-nitrophenol (m
per moles) formed per min per mg protein.
Mean OD of the untreated cells (control), is the mean of the
untreated cells used as the control.
Cellular uptake analysis by confocal microscope. MDA-MB-
231 cells were cultured in 96-well plates and incubated with
PTX- and 7-HC-loaded SLN for 24 h. Cells were washed three
times with PBS, xed with 5% paraformaldehyde for 30 min,
and stained with 4,6-diamidino-2-phenylindole (DAPI; 1 mg
mLꢁ1 in PBS) for 10 min. Cells were examined by confocal laser
scanning microscopy (Olympus IX 81 under DU897 mode,
Japan).
Animals and experimental design. Female Sprague-Dawley
rats (eight weeks old) were separated into four groups, with
each group having six animals. The regimen dose is shown in
Table 1. All groups of animals had taken food and water, which
was monitored daily for 14 days. A permanent amount of rat
chow and uid was given to every rat and relled aer that day.
Aer treatment periods of 6 weeks, the rats were fasted for two
nights, anesthetized and sacriced by cervical decapitation.
Whole blood samples were collected from both separations like
plasma/serum, respectively. The mammary tissue was cut and
washed in ice-cold saline and weighed. Tissue was cut up and
homogenized with 0.1 M Tris–HCl buffer (pH 7.4, and 10%) and
centrifuged (3000 ꢃ g for 10 min). The nally obtained super-
natant solution was used for enzyme assays. The bodyweights of
the whole animals were recorded before and aer the treatment
and sacrice.
Measurement of caspase-3, -8, and -9 activities. Caspase
activation is a common apoptotic mechanism by which anti-
cancer agents induce apoptosis. Hence, the level of caspase
activation was determined for PTX- and 7-HC-loaded SLN
system. The activities of caspase-3, -8, and -9 were determined
using a caspase colorimetric assay kit according to the manu-
facturer's recommended protocol (R and D Systems Inc., USA).
Spectrophotometric detection by the chromophore p-nitro-
anilide (pNA) assay was used for the cleavage from the labeled
substrate that recognizes an optimal tetrapeptide sequence of
the individual activation sites. Briey, tissue homogenates were
washed with ice-cold PBS, lysed with 50 mL of cold lysis buffer
and incubated on ice for 10 min. Protein concentrations of the
homogenates were assessed by the Bradford method, and 200
mg of protein was diluted in 50 mL lysis buffer solution. In
addition to that, 50 mL of 2ꢃ reaction buffer (containing 10 mM
DTT) and 4 mM DEVD-pNA substrate were added to each
sample well. Aer incubation at 27 ꢀC for 2 h, samples were read
at 405 nm using a microplate reader (BioTek, USA). Changes in
caspase activity were determined by comparing these results
with the level of control.
The experimental work was performed with ethical norms
approved by committee members of the Animals (CPCSEA),
Ministry of Environment and Forests (Animal Welfare Division),
Table 1 Experimental animal groups and treatment conditionsa
Group
Group name
Control
Treatment condition
Group I
Group II
Intraperitoneal injection of 1 mL saline per
week for four weeks
Mammary carcinoma induced by a single dose
of intraperitoneal injection of 25 mg per kg b.wt
of DMBA dissolved in saline
Induced
Group III
Group IV
SLN unloaded
SLN loaded
Mammary carcinoma induced same as group II
and treated with an intraperitoneal injection of
unloaded nanocarriers (5 mg per kg b.wt per
week for four-six weeks)
Mammary carcinoma induced same as group II
and treated with an intraperitoneal injection of
PTX- and 7-HC-loaded SLN (5 mg per kg b.wt per
week for six weeks)
a
b.wt, body weight; DMBA, 7,12-dimethylbenz[a]anthracene.
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Histopathological analysis. The evaluation of mammary and
liver tissues preserved in formaldehyde (10%) and embedded in
paraffin was conducted through microscopic examination. The
tissues were separated at 4–5 mm thickness via a semi-
automated microtome and consequently stained with
hematoxylin/eosin. Sections were viewed under a microscope
(20ꢃ magnication).
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reports.34 The purity of 4-hydroxyprolinebetaine and PB was
identied by HPLC with 70 : 30 and 50 : 50 ratios of ethyl
acetate/methanol solvent, respectively. The HPLC trace of the
70 : 30 ratio fractions revealed two peaks, one major peak and
one minor peak. The major peak noted at a retention time (RT)
of 25.6 corresponds to PB, and the minor peak observed at a RT
of 23.6 corresponds to 4-hydroxyprolinebetaine (ESI Fig. 2†).
These peaks were conrmed by standards and previously re-
ported literature.34 Similarly, in the ethyl acetate/methanol
50 : 50 ratio fraction, the major peak was noted at a RT of
24.9, corresponding to PB.
The secondary metabolites PB and LA were used for the
synthesis of NADESs with various ratios of 1 : 1, 1 : 2, 1 : 3, and
2 : 1. Ratios of 1 : 2 and 1 : 3 formed as a transparent solution,
and the physicochemical properties were characterized,
including the melting point and density of the solvents. NADES
was created into exact molar ratios of the components to reach
the eutectic point (Scheme 1A). NADES-based solid–liquid
polymeric nanocarrier was synthesized by the interaction of free
amine groups of the biotin-g-lysine-co-PEG polymer and –OH
groups of the PB–LA solvent. The synthetic route of the NADES-
based biotin-g-lysine-co-PEG polymer is given in Scheme 1A.
Statistical analysis
The results were analyzed using a single way of variance, and
whole results were obtained as mean ꢂ standard deviation (SD).
All experimental work was performed in triplicate. Results with
*P < 0.05 were considered statistically signicant.
Results
Synthesis of NADES-supported biotin-conjugated solid–liquid
polymer
For the formulation of the NADES, 4-hydroxyprolinebetaine and
PB were isolated from Citrus limon peels and identied by
HPLC, 1H-NMR, 13C-NMR, and FT-IR spectroscopy and the
spectroscopic results were compared with those of previous
Scheme 1 Schematic representation of (A) NADES synthesis and (B) synthetic route of the biotin-conjugated solid–liquid polymer.
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Table 2 The density of the NADESsa
and 1612 cmꢁ1 (carbonyl stretching vibrations). Fig. 1B shows
the LA moiety characteristic peaks at 3570, 1726, and 1081 cmꢁ1
corresponding to the OH, C–O, and C–C group vibration peaks.
Fig. 1C and D present the typical spectra of NADES-1:2 and
NADES-1:3 solvents. The COO–, C–O, –CH and C–C bands were
Temperature Density (g
Code Composition (ꢀC) cmꢁ3
)
Observation (RT)
1
2
3
4
PB : LA (1 : 1) 27
PB : LA (1 : 2) 27
PB : LA (1 : 3) 27
PB : LA (2 : 1) 27
1.1571
1.1425
1.1713
1.1358
Semisolid
observed at 1360 cmꢁ1, 1635 cmꢁ1, 1646 cmꢁ1, 1638 cmꢁ1, 755
Transparent liquid
Highly viscous
Semisolid
and 752 cmꢁ1
.
Broad peaks were observed at 2934 and
15,16
3322 cmꢁ1, which indicates that the hydroxyl group (OH) was
involved in strong intramolecular hydrogen bonding during
NADES formation. Also, the new characteristic peaks of NADES
conrm the interaction of LA and PB moiety. As shown in
Fig. 1E, the peaks at 3295 cmꢁ1 (O–H), 2935 cmꢁ1 (C–H),
1570 cmꢁ1 (N–H), 1378 cmꢁ1 (C–C), 1690 cmꢁ1 (C]O), and
1378 cmꢁ1 (C–O) are revealed by biotin molecule. Fig. 1F shows
a strong peak at 1666 cmꢁ1 corresponding to amide bonds and
represents the amide bond of the biotin graed lysine (biotin-g-
lysine).17,36 The FT-IR spectrum of biotin-g-lysine-co-PEG (biotin-
conjugated solid–liquid polymer) (Fig. 1G) demonstrates char-
acteristic peaks at 1596 and 1726 cmꢁ1, which represent the
amide and ester stretching vibration of PEG and biotin-g-lysine,
respectively.37 The characteristic peaks at 1095, 1241, 1513,
1560, 1651, 1734, 2940, and 3376 cmꢁ1 show a strong hydro-
phobic interaction between nanocarriers and drugs (PTX and 7-
HC). All the characteristic peaks conrm the synthesis of PTX-
and 7-HC-loaded NADES-based biotin-conjugated solid–liquid
polymer nanocarrier (Fig. 1H).
a
PB, prolinebetaine; LA, lactic acid; RT, room temperature.
Density analysis
Solvent density plays a critical role in chemical synthesis, and it
is an essential physical property. In drug carrier synthesis using
ILs, DES, and NADES, density plays a signicant role in the
formation of drug delivery systems (DDSs).29 The densities of
the NADES-1 : 1, NADES-1 : 2, NADES-1 : 3, and NADES-2 : 1
solvents are mentioned in Table 2. The NADES density values
ranged from 1.1571 to 1.1713 g cmꢁ3 at room temperature
(27 ꢀC). NADES-1:3 had the maximum density (1.1713 g cmꢁ3) at
27 ꢀC. As the concentration of LA increased, simultaneously the
molecular kinetic energy also increased because of the vibration
of the atoms which were available in the NADES. It is seen that
since the mobility of the NADES improved, the densities of the
solvent decreased.
NMR analysis
XRD, Raman spectroscopy and TGA
The interaction of the PB : LA-based NADES formation was
The XRD patterns and Raman spectra of biotin-g-lysine-co-PEG
polymer, NADES-based biotin-conjugated solid–liquid polymer,
and PTX- and 7-HC-loaded nanocarrier were studied. The XRD
pattern of biotin-conjugated solid–liquid polymer nanocarrier
exhibited peaks at 2q values of 23.11, 25.6, 26.5, 27.8, 29.9,
31.56, 43.90, 44, 49.32, 50.1, 53.5 and 56.0ꢀ, indicating the
graed polymer is semi-crystalline (Fig. 2a(A)). As shown in
Fig. 2a(B), the intensity of the characteristic peaks was
decreased due to the interaction between NADES biotin-g-
lysine-co-PEG polymer in the formation of SLN, and the 2q
values are 15.41, 22.47, 25.71, 31.70, and 45.31ꢀ. PTX shows
multiple peaks at 2q values of 23, 35, 39.01, 41.25, 44.56, 47.21,
and 66ꢀ due to its crystalline nature (Fig. 2a(C)).38 The XRD
pattern of 7-HC exhibited peaks appearing at 2q values of 28.15,
30.15, 31.39, 32.20, 33.83, 44.37, 46.00, 54.92, 62.23, 65.83,
67.52, and 69.97ꢀ (Fig. 2a(D)).39 Aer encapsulation of both
drugs in the SLN system, the XRD pattern shows broad, less
intense peaks, with a minor shi. This indicates that the drug-
encapsulating SLN system has excellent interactions with
a semi-crystalline nature (Fig. 2a(E)). Generally, a semi-
crystalline system has a good interaction with biological
liquids, which may be tting for penetration through tissue
layers and might provide an excellent diffusion property for the
1
examined by using H- and 13C-NMR to conrm the structure.
1
The HBA of PB characteristic signal of H values: 4.23 (t, CH),
3.05 (s, 2CH3), 3.00 (q, CH2), 2.01 (p, CH2), 1.83 (q, CH2) and 13
C
NMR values: 185, 93, 55, 43, and 28 (ESI Fig. 3†). The HBD of LA
showed 1H NMR values: 4.67 (s, OH), 4.11 (s, OH), 1.47 (d, 3H),
1.11 (1, 1H), and 13C NMR values: 185, 70, 25 (ESI Fig. 4†). Aer
1
the interaction of PB : LA (NADES-1:2) H values: 10.73 (s, 1H),
5.21 (q, CH2), 4.21 (q, CH2), 3.36 (s, 2CH2), 3.42 (q, CH2), 2.56 (s,
1H), 1.81 (p, CH2), 1.79 (p, CH2), 0.8 (s, CH2) and 13C NMR
values: 170, 89, 77, 75, 58, 28, and 26 (ESI Fig. 5†). In ESI Fig. 6,†
the formation of NADES-1:3 characteristic signal of the 1H NMR
values: 10.71 (s, 1H), 6.51 (q, CH2), 5.51 (q, CH2), 4.45 (s, 2CH2),
4.01 (q, CH2), 4.00 (s, 1H), 2.71 (p, CH2), 2.62 (p, CH2), 2.01 (s,
CH2) and 13C NMR values: 173, 93, 79, 78, 56, 27, and 24. The
spectral results show that the hydrogen bonding of (O–H+/ꢁO)
group is indicative of interaction with HBA and HBD in NADES
1
formation (Fig. S2C†). The characteristic H signals of NADES
were shied downeld when compared with free PB and LA,
which is a result of excellent hydrogen bond interaction of
NADES.35
FT-IR analysis
PB, LA, NADES, biotin-g-lysine-co-PEG polymer, and PTX- and 7- nanocarriers with drug molecules. Since the nature of the PTX-
HC-loaded NADES-based biotin-conjugated solid–liquid poly- and 7-HC-loaded SLN system allows for interaction with the cell
mer nanocarrier were characterized by FT-IR spectroscopy. The membranes, these materials have been used.
results are shown in Fig. 1. Fig. 1A shows the absorption bands
Raman spectra were investigated to study the interaction
of PB functional groups at 1355 cmꢁ1 (–CH stretching vibration) between PTX and 7-HC and SLN (Fig. 2b). The bands at
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Fig. 1 FT-IR spectra of (A) prolinebetaine, (B) lactic acid, (C) NADES-1:2, (D) NADES-1:3, (E) biotin, (F) biotin-g-lysine, (G) biotin-g-lysine-co-PEG,
and (H) PTX- and 7-HC-loaded NADES-based biotin-conjugated solid–liquid polymer (PTX- and 7-HC-loaded nanocarrier).
1126 cmꢁ1 are assigned to the C–N stretching vibration mode of degradation occurs as a multistep process compared with the
PB, and the band at 865 cmꢁ1 is attributed to the strong C–COO nanocarrier alone. Thermal stability is a powerful force of
bending mode.26 The bands at 1319 cmꢁ1, 1371 cmꢁ1, and molecules for bond energy and interactions. The TGA curve
1069 cmꢁ1 are attributed to the strong CH3 stretching mode. shows a slight decrease in the range of 25–35 ꢀC and corre-
The band at 1680 cmꢁ1 was assigned to the ester vibration sponds to the elimination of moisture in the nanocarrier
mode.40 The characteristic vibration bands at 1680 cmꢁ1 cor- (Fig. 2c(A)). The 2nd stage of weight reduction is from 150 to
responded to the amide stretching vibration modes, as shown 165 ꢀC due to CO and CO2,41 and the third stage of weight loss
in Fig. 2b(A). Fig. 2b(B) shows the Raman spectrum of PTX- and from 320 to 350 ꢀC is related to the decomposition of ester and
7-HC-loaded nanocarrier and vibration bands appeared at 278, amide linkages. In contrast, from the weight loss of PTX- and 7-
293, 309, 340, 366, 392, 926, 952, and 1066 cmꢁ1. This reveals HC-loaded nanocarrier, it was found to have a higher thermal
the durable hydrophobic nature and hydrophobic interaction stability compared to the unloaded nanocarrier. At a tempera-
between nanocarrier and drugs.
ture of 210 ꢀC, the mass had reduced by 85% in the carrier,
The thermal stability and decomposition of the unloaded followed by a gradual reduction of weight at 400–500 ꢀC
and PTX- and 7-HC-loaded nanocarrier were characterized by (Fig. 2c(B)).
TGA. Fig. 2c reveals PTX- and 7-HC-loaded nanocarrier
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Fig. 2 (a) XRD patterns of (A) biotin-conjugated solid–liquid polymer, (B) NADES-based biotin-conjugated solid–liquid polymer, (C) PTX, (D) 7-
HC, and (E) PTX- and 7-HC-loaded nanocarrier. (b) Raman spectra of (A) nanocarrier and (B) PTX- and 7-HC-loaded nanocarrier. (c) TGA
measurements for (A) nanocarrier and (B) PTX- and 7-HC-loaded nanocarrier. (d) Fluorescence spectra of micelles (A) intensity ratio I1/I3 of
pyrene as a function of nanocarrier concentration, and (B) the CMC in an aqueous medium is determined to be about 0.6 mg mLꢁ1
.
conrmed the amphiphilic polymer changes (Fig. 2d). The CMC
value of NADES-based biotin-conjugated solid–liquid polymer
nanocarrier was identied in the aqueous medium and
Determination of self-assembly of carrier formation
The assistance of NADES enabled the synthesis of the NADES-
based biotin-conjugated solid–liquid polymer nanocarrier
through self-assembly. Here, the biotin-conjugated solid–liquid
polymer nanocarrier acted with hydrophilic character and
NADES with hydrophobic character, and it was a self-assembled
from a solid–liquid polymeric system by dialysis. The self-
assembling of the NADES-based biotin-conjugated solid–
liquid polymer system was conrmed through critical nano-
carrier concentration using pyrene as a uorescent probe. The
rst and third shi of the NADES-based biotin-conjugated
solid–liquid polymer nanocarrier (0.1 mg mLꢁ1) was found in
the range of I390/I445, respectively. The nal concentration of the
NADES-based biotin-conjugated solid–liquid polymer nano-
carrier (0.9 mg mLꢁ1) was observed at I377/I442. The shiing of
the higher region to lower one by changing the concentrations
observed at 0.6 mg mLꢁ1
.
Surface morphological analysis
The size determination and morphological characterization of
the nanocarrier were conducted by SEM and TEM instru-
mental techniques (Fig. 3a). The surface feature of the carrier
system can enhance the bio-distribution and pharmacoki-
netics of drugs in biological systems, and it has a vital role in
the treatment of the diseases.42 Fig. 3a(A) shows that the
nanocarrier is spherical, its size is increased with the incor-
poration of drugs and it is semi-crystalline. The SLN particle
observed in the SEM image showed that the NADES liquid was
covering to stabilize of the solid particle, and the solid portion
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Fig. 3 (a) SEM images of (A) nanocarrier and (B) PTX- and 7-HC-loaded nanocarrier. TEM images of (C) nanocarrier and (D) PTX- and 7-HC-
loaded nanocarrier. The blue arrow indicates the hydrophilic outer layer, and the yellow arrow indicates the hydrophobic inner layer. (b) Zeta
potential of (A) nanocarrier and (B) PTX- and 7-HC-loaded nanocarrier, and particle size of (C) nanocarrier and (D) PTX- and 7-HC-loaded
nanocarrier.
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was highly dense in the drug-loaded SLN system (Fig. 3a(B)). at ꢁ3 ꢂ 0.5 mV and ꢁ0.2 ꢂ 0.5 mV for unloaded and PTX- and 7-
Furthermore, the PTX- and 7-HC-loaded nanocarrier was HC-loaded SLN systems, respectively. From these observations,
spherical, and the size of the particle increased due to the drug-loaded SLN had enhanced stability over the unloaded
charged drug molecules. Fig. 3a(C and D) show representative carrier, due to the interaction of the drugs and carrier mole-
TEM images of the nanocarrier, the particles of which are cules. The observed zeta potential was nearly zero (Fig. 3b(A and
spherical with a mean particle size of 200–300 nm; this B)). The surface charge of the nanocarrier has a meaningful
correlates well with the SEM images.
relevance for biological applications for stability, interaction
with cells, and also tissues.43 The size distribution of the
nanocarrier was observed in the range of z240 nm for the
unloaded and z290 nm for the loaded carrier. This indicated
Particle size analysis and zeta potential determinations
The surface charge and particle size of the unloaded and PTX- that molecule size expanded as PTX and 7-HC were incorpo-
and 7-HC-loaded SLN system were tested using DLS, and the rated in the nanocarrier (Fig. 3b(C and D)).
results are presented in Fig. 3b. The zeta potential was observed
Fig. 4 UV-visible spectroscopy showing (A) encapsulation efficiency and (B) percentage encapsulation efficiency of PTX- and 7-HC-loaded
nanocarrier. In vitro drug discharge status of PTX- and 7-HC-loaded nanocarrier at (C) pH 2.8, (D) pH 5.5, and (E) pH 7.4. (F) Cumulative drug
release pattern over 320 min.
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Fig. 5 (A) The cell viability of PTX- and 7-HC-loaded SLN-treated normal cell line. (B) The cytotoxicity of PTX- and 7-HC-loaded SLN against
MDA-MB-231 cancer cell line.
In vitro release
In vitro cell cytotoxicity
The in vitro drug release proles of anticancer drugs discharged The cytotoxic effect of SLN systems was estimated using
from the nanocarrier were investigated for solutions at different a normal broblast cell line (L929) and a breast cancer cell line
pH by UV-visible spectroscopy (Fig. 4). The variation of drug (MDA-MB-231) through the MTT assay for 24 h with various
release at different pH indicates that the carrier has pH- concentrations. The treatment of unloaded SLN and PTX- and 7-
dependent drug release. At an acidic pH, a burst in drug HC-loaded SLN system retains cell viability in the normal
release was observed when compared with other pH values. At broblast cell line (ESI Fig. 8A†), with quantitative cell viability
pH 2.8, 27.47% drug release was observed in the rst 30 min. On being shown in Fig. 5A. ESI Fig. 8B† shows the viability of MDA-
the other hand, at pH 7.4, only 8.79% was released from the MB-231 cells treated with unloaded and PTX- and 7-HC-loaded
carrier. The drug was released from the nanocarrier for up to SLN. Viability decreased at a concentration of 100 mg mLꢁ1, i.e.,
320 min, with a maximum drug release at an acidic pH of 2.8 increased cell death was noticed when compared with the
(97.0%) compared with a pH of 7.4 (89%) and pH 5.5 (92%) nanocarrier at lower concentrations; the cell cytotoxicity value
within 320 min. Burst releases followed by the increasing drug of various concentrations is presented in Fig. 5B.
discharge pattern of the drugs from the carrier in a strongly
acidic solution could be due to the amine group and ester easily
protonating the nanocarrier in a strongly acidic environment
Cellular uptake studies
compared with a nearly neutral pH solution.
MDA-MB-231 breast cancer cells were cultured and treated with
the PTX- and 7-HC-loaded SLN system for 24 h (Fig. 6).
Fig. 6 Confocal images of MDA-MB-231 cancer cells incubated with PTX- and 7-HC-loaded SLN system at 27 ꢀC for (A) 1 h, (B) 5 h, (C) 10 h, (D)
15 h, (E) 20 h, and (F) 24 h. White arrows indicate the presence of PTX- and 7-HC-loaded SLN around nuclei.
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Fluorescence images were observed aer 1 h, 5 h, 10 h, 15 h,
20 h, and 24 h. As the treatment time increased, uorescent
intensities increased, which is due to a higher uptake of PTX-
and 7-HC-loaded carriers via receptor-mediated endocytosis.
The uorescence signal was very strong, and co-localization
around the nucleus (white arrow) was observed at 20 h and
24 h in the PTX- and 7-HC-loaded SLN system-treated samples.
Histological studies. The histological sections of breast
tissue and liver tissue of DMBA-induced breast cancer in rats
treated with 5 mg per g b.wt of PTX- and 7-HC-loaded SLN are
shown in Fig. 8. In both the liver and the mammary tissue
sections, the loaded SLN-treated animals showed normal and
healthy architecture when compared to the other groups.
Discussion
In vivo studies
The SLN size was chosen based on tissue penetration, pro-
Lysosomal enzymes. Fig. 7A presents the levels of lysosomal longed persistence in the bloodstream thereby avoiding renal
marker enzymes such as acid phosphatase, b-D-glucosamini- ltration, and also better effectiveness for cancer therapy. As
dase, and cathepsin-D in plasma and mammary tissue of a result, the swelling behavior increased as the pH decreased.
control and treated rats.
Moreover, the NADES-based biotin-conjugated solid–liquid
Effects of PTX- and7-HC-loaded SLN on caspase-3, -8 and -9 polymer, aer arriving at the targeted cancer cells, will be
activities. To elucidate cell death induced by PTX- and 7-HC- degraded by lysosomal enzymes that are abundant in cancer
loaded SLN, the possible involvement of caspases was cells. The primary role of these enzymes is the degradation of
assessed (Fig. 7B).
ester and amide bonds. In this system, the drug carrier has both
Fig. 7 (B and C) Effect of PTX- and 7-HC-loaded nanocarrier on lysosomal marker enzymes. Group I, control; group II, cancer induced; group III,
SLN unloaded treated; group IV, SLN loaded treated. Each value is expressed as the mean ꢂ SD for six rats in each group. Significance compared
to group I is shown as *P < 0.05. (A) Effect of PTX- and 7-HC-loaded nanocarrier on caspase-3, -8, and -9. Group I, control; group II, cancer
induced; group III, SLN unloaded treated; group IV, SLN loaded treated. Each value is expressed as the mean ꢂ SD for six rats in each group.
Significant difference as compared to group I shown as *P < 0.05.
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Fig. 8 Histological sections of mammary tissue of DMBA-induced breast cancer in rats from (A) control, (B) cancer induced, (C) SLN unloaded
treated, and (D) SLN loaded treated. Liver sections of DMBA-induced breast cancer in rats from (E) control, (F) cancer induced, (G) SLN unloaded
treated, and (H) SLN loaded treated.
amide and ester bonds. The amide bonds found in the NADES signicantly elevated (plasma and mammary tissue (P < 0.05)) in
will allow the lysosomal enzymes to simply degrade the biotin- group II animals when compared with the controls. This may be
conjugated solid–liquid polymer in cancer cells.41 Thus, it will due to the abnormal release of lysosomes in cancer conditions.
help the release of drugs into cancer cells. The cell cytotoxicity Also, the elevated levels could reect the increased synthesis
effect of the PTX- and 7-HC-loaded SLN is due to a high negative and secretion of lysosomal enzymes by tumors.35 Due to the
surface charge that eases the binding on the surface of cancer treatment with PTX- and 7-HC-loaded SLN system in group IV
cells, thereby damaging the cell walls and altering adenosine animals, the enzyme levels decreased to near normal.
triphosphate (ATP) production. Cell wall damage is the most
In most cases, different anticancer agents eventually mediate
signicant determinant of the cytotoxic activity due to the a common apoptotic pathway through the activation of cas-
strong electrostatic interaction involving the negatively charged pases. The enzymatic activity of caspase-3, -8, and -9 in the PTX-
polymer carrier and the positively charged cell membrane.44 and 7-HC-loaded SLN-treated rats increased signicantly.
Based on the cell uptake properties of the drug-loaded poly- Otherwise, group II is a cancer-induced cell line. Aer that, the
meric micelles, this suggests that PTX and 7-HC present in the induced cell line was incubated with polymeric micelles, which
nanocarrier interact with DNA and damage the nuclear gradually increased in group III due to the SLN's functional
membrane of cancer cells.38 From this, it could be seen that group activity in the cancer cell line. On the other hand, the
there are changes in the tumor volume and body weight, which caspase levels in the SLN-treated group remain unchanged. This
are shown in Table 3. In cancer rats, the body weight was reects that the treatment with PTX- and 7-HC-loaded SLN leads
decreased (P < 0.05) as compared to the control rats. In PTX- and to a strong cleavage of the biotin targeted molecules. However,
7-HC-loaded SLN system-treated rats, the body weight was the targeted drug delivery of PTX- and 7-HC-charged SLN to
notably recovered and also the tumor size signicantly reduced tumor cells excellently enhanced the apoptosis-induced cell
as compared with the cancer-induced and unloaded death. The histological sections of PTX- and 7-HC-loaded SLN-
nanocarrier-treated rats. The lysosomal enzyme activities were treated rats reveal the presence of atrophy of seromucous
Table 3 Effect of nanocarriers on body weight and tumor volume in control and experimental ratsa
Body weight
Group I
Group II
Group III
Group IV
Initial
Final
199.5 ꢂ 8.26
239.5 ꢂ 4.33
—
197.8 ꢂ 2.99
210.1 ꢂ 3.98*
4502 ꢂ 380*
198.6 ꢂ 5.39
212.7 ꢂ 4.85*
4289 ꢂ 403*
195.6 ꢂ 5.81
243.2 ꢂ 9.21
2954 ꢂ 335
Tumor volume
(mm2)
a
Group I, control; group II, cancer induced; group III, SLN unloaded treated; group IV, SLN loaded treated. Each value is expressed as the mean ꢂ
SD for six rats in each group. Signicance as compared to group I shown as *P < 0.05.
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glands with surrounding stromal brosis, fatty tissue with small
lobules, and tumor regression. DMBA-induced rats showed
tumors composed of hyperchromatic, pleomorphic, vesicular
nuclei, and moderate cytoplasm arranged in nests, sheets, and
acinar structures with numerous mitotic structures. Based on
the results, the PTX- and 7-HC-loaded SLN can reduce side
PB
LA
b.wt
DMBA
DDSs
ILs
Prolinebetaine
Lactic acid
Bodyweight
7,12-Dimethylbenz[a]anthracene
Drug delivery systems
Ionic liquids
effects. Ultimately, histological studies of the SLN and PTX- and DESs
Deep eutectic solvents
Double-distilled water
Adenosine triphosphate
Retention time
DD water
7-HC-loaded SLN did not reveal any abnormal morphological
change to the mammary tissue and liver, further showing that
they are safe for therapeutic use due to the PEG corona pro-
tecting against protein absorption with highly stable SLN and
PTX- and 7-HC-loaded SLN, while the PEGylated surface led to
an uptake in the liver.
ATP
RT
Conflicts of interest
The authors declare that they have no competing interests.
Conclusion
A novel approach in advanced cancer prevention and treat-
ment includes the use of SLN systems for anticancer drug
delivery. A PTX- and 7-HC-loaded SLN was synthesized for the
rst time with a green solvent as a promising novel drug
delivery system for the enhancement of the therapeutic index
of cancer cells. The unloaded and PTX- and 7-HC-loaded SLNs
were characterized in terms of size and crystalline properties.
The higher toxicity of PTX- and 7-HC-loaded SLN system was
observed by MTT assay, which promoted MDA-MB-231 cell
death. The effect of PTX- and 7-HC-loaded SLN on cell viability
was investigated with MTT assay and also by morphological
observations. The decreased cell viability observed in this
study could be due to direct inhibition of cell growth by PTX-
and 7-HC-loaded SLN. Apoptosis was included in cell death
mechanisms observed in breast cancer-induced rats. Treat-
ment with PTX- and 7-HC-loaded SLN system elevates caspase
levels that stimulate cell death mechanisms and damage
tumor tissue. From these results, it is clear that the synthe-
sized PTX- and 7-HC-loaded SLN system is highly valuable in
preventing cancer cell proliferation. It is concluded that the
PTX- and 7-HC-packed SLN system might be a potential
candidate for breast cancer treatment and also which can
improve the treatment of incurable diseases. Additional
studies are suggested for estimating the effect of these nano-
carriers on other cell death mechanisms.
Acknowledgements
M. Rajan is grateful for nancial support under the plan of
“ICMR” (F. No. 5/3/8/350/2018-ITR; New Delhi, India) and
Department of Science and Technology, Science and Engi-
neering Research Board (Ref: YSS/2015/001532; New Delhi,
India) and also acknowledges the PURSE program for the
purchase of SEM and FT-IR and UPE programs for the purchase
of TEM. The authors extend their appreciation to the
researchers supporting project number RSP-2020/190, King
Saud University, Riyadh, Saudi Arabia.
References
1 J. Brindha and C. Kaushik, Evolutionary approaches in
protein engineering towards biomaterial construction, RSC
Adv., 2019, 9, 34720–34734.
2 S. Mehnath, M. Arjama, M. Rajan, K. Premkumar,
K. Karthikeyan and M. Jeyaraj, Mineralization of bioactive
marine sponge and electrophoretic deposition on Ti-6Al-4V
implant for osteointegration, Surf. Coat. Technol., 2020,
392, 125727.
3 F. Khan, M. Tanaka and S. R. Ahmad, Fabrication of
Polymeric Biomaterials: A Strategy for Tissue Engineering
and Medical Devices, J. Mater. Chem. B, 2015, 3, 8224–8249.
4 H. H. Tonnesen and J. Karlsen, Alginate in Drug Delivery
Systems, Drug Dev. Ind. Pharm., 2002, 28, 621–630.
5 S. Palvai, L. Anandi, S. Sarkar, M. Augustus, M. Roy, M. Lahiri
and S. Basu, Drug-Triggered Self-Assembly of Linear Polymer
into Nanoparticles for Simultaneous Delivery of
Hydrophobic and Hydrophilic Drugs in Breast Cancer
Cells, ACS Omega, 2017, 2, 8730–8740.
Abbreviations
NADES
SLN
NMR
FTIR
TGA
XRD
DLS
SEM
TEM
PTX
Natural deep eutectic solvents
Solid–liquid nanocarrier
Nuclear magnetic resonance
Fourier-transform infrared spectroscopy
Thermogravimetric analysis
Powder X-ray diffraction
Dynamic light scattering
Scanning electron microscopy
Transmission electron microscopy
Paclitaxel
6 D. Govindaraj, P. Pradeepkumar and M. Rajan, Synthesis of
morphology tuning multi mineral substituted apatite
nanocrystals by novel natural deep eutectic solvents, Mater.
Discovery, 2018, 9, 11–15.
7 N. Graf and S. J. Lippard, Redox Activation of Metal-Based
Prodrugs as A Strategy for Drug Delivery, Adv. Drug Delivery
Rev., 201, 264, 993–1004.
7-HC
7-Hydroxycoumarin
8 Y. Ishihara and N. Shimamoto, Involvement of
MDA-MB-231 M. D. Anderson and metastasis breast cancer
Endonuclease
G in Nucleosomal DNA Fragmentation
37002 | RSC Adv., 2020, 10, 36989–37004
This journal is © The Royal Society of Chemistry 2020

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Paper
RSC Advances
under Sustained Endogenous Oxidative Stress, J. Biol. Chem., 21 S. S. Heinzmann, I. J. Brown, Q. Chan, M. Bictash,
2006, 281, 6726–6733.
M. E. Dumas, S. Kochhar and J. Stamler, Metabolic
proling strategy for discovery of nutritional biomarkers:
prolinebetaine as a marker of citrus consumption, Am. J.
Clin. Nutr., 2010, 92, 436–443.
9 S. G. Arguelles, M. C. Serrano, M. C. Gutieerrez, M. Luisa
Ferrer, L. Yuste, F. Rojo and F. del Monte, Deep Eutectic
Solvents for the Self-Assembly of Gold Nanoparticles: A
SAXS, UVꢁVis, and TEM Investigation, Langmuir, 2013, 29, 22 M. J. Joralemon, K. S. Murthy, E. E. Remsen, M. L. Becker
9525–9534.
and K. L. Wooley, Synthesis, Characterization, and
Bioavailability of Mannosylated Shell Cross-Linked
Nanoparticles, Biomacromolecules, 2004, 5, 903–913.
10 H. T. Thai Thanh, H. P. Emily, N. Dai Hai, S. L. Jung, P. Ki
Dong and P. T. Nghia, The Importance of Poly(ethylene
glycol) Alternatives for Overcoming PEG Immunogenicity 23 J. H. Kim, Y. Li, M. S. Kim, S. W. Kang, J. H. Jeong and
in Drug Delivery and Bioconjugation, Polymers, 2020, 12,
298.
11 S. Ksenia, S. Egorova, G. Evgeniv, G. Gordeev and
D. Sung Lee, Synthesis and Evaluation of Biotin-
Conjugated Ph-Responsive Polymeric Nanocarriers as Drug
Carriers, Int. J. Pharm., 2012, 427, 435–442.
P. Valentine, Biological Activity of Ionic Liquids and Their 24 Y. Miyake, K. Yamamoto, Y. Morimitsu and T. Osawa,
Application in Pharmaceutics and Medicine, Chem. Rev.,
2017, 117, 7132–7189.
12 P. Pradeepkumar, A. M. Elgorban, A. H. Bahkali and
Isolation of C-Glucosylavone from Lemon Peel and
Antioxidative Activity of Flavonoid Compounds in Lemon
Fruit, J. Agric. Food Chem., 1997, 45(12), 4619–4623.
M. Rajan, Natural Solvent-Assisted Synthesis of 25 V. S. Raghuwanshi, M. Ochmann, A. Hoell, F. Polzer and
Amphiphilic Co-Polymeric Nanonanocarrier for Prolonged
Release of Camptothecin Delivery, New J. Chem., 2018, 42,
10366–10375.
K. Rademann, Deep Eutectic Solvents for the Self-Assembly
of Gold Nanoparticles:
SAXS, UVꢁVis, and TEM
Investigation, Langmuir, 2014, 30, 6038–6046.
A
13 R. Verpoorte, Chemodiversity and the Biological Role of 26 W. Zhu, Z. Song, P. Wei, N. Meng, T. Teng, F. Yang, N. Liu
Secondary Metabolites, Some Thoughts for Selecting Plant
Material for Drug Development, in Bioassay Methods in
Natural Product Research and Drug Development, ed. L.
Bohlin and J. G. Bruhn, Proceedings of the Phytochemical
and R. Feng, Y-Shaped Biotin-Conjugated Poly (Ethylene
Glycol)–Poly (Epsilon-Caprolactone) Copolymer for The
Targeted Delivery of Curcumin, J. Colloid Interface Sci.,
2015, 443, 1–7.
Society of Europe, Springer, Dordrecht, 1999, vol. 43, pp. 27 A. S. Mikhail and C. Allen, (Poly(ethylene glycol)-b-poly(3-
11–23.
caprolactone))
Nanocarriers
Containing
Chemically
14 W. Czaban, J. Rasmussen, B. B. Laursen, N. H. Vidkjaer,
R. Sapkota, M. Nicolaisen and S. Fomsgaard, Multiple
Effects of Secondary Metabolites on Amino Acid Cycling in
White Clover Rhizosphere, Soil Biol. Biochem., 2018, 123,
54–63.
15 S. Ksenia, S. Egorova, G. Evgeniv, G. Gordeev and
P. Valentine, Biological Activity of Ionic Liquids and Their
Application in Pharmaceutics and Medicine, Chem. Rev.,
2017, 117, 7132–7189.
Conjugated and Physically Entrapped Docetaxel: Synthesis,
Characterization, and the Inuence of the Drug on
Nanocarrier Morphology, Biomacromolecules, 2010, 11,
1273–1280.
28 R. A. Praphakar, M. A. Munusamy, A. A. Alarfaj, S. Suresh
Kumar and M. Rajan, Zn2+ Cross-Linked Sodium Alginate-
G-AllylamineMannose Polymeric Carrier on Rifampicin for
Macrophage Targeting Tuberculosis Nanotherapy, New J.
Chem., 2017, 41, 11324–11334.
16 P.
Pradeepkumar,
D.
Govindaraj,
M.
Jeyaraj, 29 P. Pradeepkumar, R. Naresh Kumar, A. A. Alarfaj,
M. A. Munusamy and M. Rajan, Assembling of
Multifunctional Latex-Based Hybrid Nanocarriers from
Calotropisgigantea for Sustained (Doxorubicin) DOX
Releases, Biomed. Pharmacother., 2017, 87, 461–470.
M. A. Murugan and M. Rajan, Deep Eutectic Solvent-
Mediated FA-g-b-Alanine-co-PCL Drug Carrier for
Sustainable and Site-Specic Drug Delivery, ACS Appl. Bio
Mater., 2018, 1(6), 2094–2109.
17 C. Mukesh, J. Bhatt and K. Prasad, Preparation of 30 C. Janas, Z. Mostaphaoui, L. Schmiederer, J. Bauer and
a
NoncytotoxicHemocompatible Ion Gel by Self-
M. G. Wacker, Novel Polymeric Nanocarriers for Drug
Delivery: Material Characterization and Formulation
Screening, Int. J. Pharm., 2016, 509, 197–207.
Polymerization of HEMA in a Green Deep Eutectic Solvent,
Macromol. Chem. Phys., 2014, 215, 1498–1504.
18 A. L. Wang, X. L. Zheng, Z. Z. Zhao, C. P. Li and X. F. Zheng, 31 J. King, The hydrolases–acid and Alkaline Phosphatises, in
Deep Eutectic Solvent Catalyzed friedel–Cras Alkylation of
Electron-Rich Arenes with Aldehydes, RSC Adv., 2014, 5,
59022–59026.
Practical Clinical Enzymology, ed. J. C. King, D. Van
Nostrand Company Ltd., London, 1965, pp. 191–208.
32 A. I. Sapolsky, R. D. Atlman, D. S. Howell and D. Cathepsin,
Activity in Normal and osteoarthritic human cartilage, Fed.
Proc., 1973, 32, 1489–1493.
19 D. Monleon, J. M. Morales, A. Barrasa, J. A. Lopez, C. Vazquez
and B. Celd, Metabolite Proling of Fecal Water Extracts
From Human Colorectal Cancer, NMR Biomed., 2009, 22, 33 Y. Kawai and K. Anno, Mucopolysaccharide Degrading
342–348.
Enzymes
from
the
Liver
of
Squid
20 C. R. Day and S. A. Kempson, Betaine Chemistry, Roles, and
Potential Use in Liver Disease, Biochim. Biophys. Acta, Gen.
Subj., 2016, 1860(6), 1098–1106.
OmmastrephesSolaniPacicus. I. Hyaluronidase, Biochim.
Biophys. Acta, Enzymol., 1971, 242, 428–436.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 36989–37004 | 37003

View Article Online
RSC Advances
Paper
34 L. Servillo, N. D'Onofrio, A. Giovane, R. Casale, D. Cautela, 40 W. C. Harris and D. A. Coe, Vibrational spectra and structure
G. Ferrari and D. Castaldo, The Betaine Prole of Cereal
Flours Unveils New And Uncommon Betaines, Food Chem.,
2018, 239, 234–241.
of esters–H.* Raman spectra and potential function
calculations for HC00CH3, DCOOCH3 and HCOOCD3,
Spectrochim. Acta, Part A, 1976, 32, 1–10.
35 H. Shinar, M. D. Battistel, M. Mandler, F. Lichaa and 41 H. Gheybi and A. A. Entezami, NanosizedNanocarriers Self-
F. L. Freed berg, Chemical Exchange Saturation Transfer
NMR of Oligo- and Poly-Sialic Acids and the Assignment of
Their Hydroxyl Groups Using Selective- and HSQC-TOCSY,
Carbohydr. Res., 2014, 389, 165–173.
assembled from Amphiphilicpoly(citric acid)–poly(e-
caprolactone)–poly(citric acid) Copolymers, Polym. Bull.,
2013, 70, 1875–1894.
42 S. S. Davis, Biomedical Applications of Nanotechnology-
Implications for Drug Targeting and Gene Therapy, Trends
Biotechnol., 1997, 15, 217–224.
36 A. L. Wang, X. L. Zheng, Z. Z. Zhao, C. P. Li and X. F. Zheng,
Deep Eutectic Solvent Catalyzed friedel–Cras Alkylation of
Electron-Rich Arenes with Aldehydes, RSC Adv., 2014, 5, 43 K. Song Yang, N. V. Mai, H. P. Emily, P. R. J. Angus,
59022–59026.
R. W. Michael, F. Q. John, P. T. Nghia and P. D. Thomas,
Elucidating the Inuences of Size, Surface Chemistry, and
Dynamic Flow on Cellular Association of Nanoparticles
Made by Polymerization-Induced Self-Assembly, Small,
2018, 14(34), 1801702.
37 C. Chien, T. Ching, A. Lin, M. Zhang, S. L. Levengood and
M. Zhang, PEG-Chitosan Hydrogel with Tunable Stiffness
for Study of Drug Response of Breast Cancer Cells,
Polymers, 2016, 8, 112.
38 H. Zhang, H. Hu, H. Zhang, W. Dai, X. Wang, X. Wang and 44 M. Rajan, P. Krishnan, P. Pradeepkumar, M. Jeyanthinath,
Q. Zhang, Effects of Pegylated Paclitaxel Nanocrystals On
Breast Cancer and Its Lung Metastasis, Nanoscale, 2015, 7,
10790–10800.
M. Jeyaraj, M. P. Ling, P. Arulselvan, A. Higuchi,
M. A. Munusamy, R. Arumugam, G. Benelli, K. Murugan
and S. Suresh Kumar, Magneto-chemotherapy for cervical
39 G. A. Shtukenberg, Q. Zhu, D. J. Carter, L. Vogt, J. Hoja,
E. Schneider and H. Song, Powder Diffraction and Crystal
Structure Prediction Identify Four New Coumarin
Polymorphs, Chem. Sci., 2017, 8, 4926–4940.
cancer
treatment
with
camptothecin
loaded
Fe3O4functionalizedb-cyclodextrinnanovehicles, RSC Adv.,
2017, 7, 46271.
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