Formulation and in vitro evaluation of quercetin loaded carbon nanotubes for Cancer Targeting

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Formulation and in vitro evaluation of quercetin loaded carbon
nanotubes for Cancer Targeting
Zahra Alimohammadi1, Asma Zamansani2, Azadeh Abreshteh3, Mehrsa Edalaat4, Zohreh
Ebrahimi5
1Faculty of Chemical Engineering, Naghshe Jahan University, Isfahan 8199874941, Iran;
2 Faculty of Chemical Engineering, Naghshe Jahan University, Isfahan 8199874941, Iran
3 Faculty of Chemical Engineering, Naghshe Jahan University, Isfahan 8199874941, Iran
4Faculty of Chemical Engineering, Naghshe Jahan University, Isfahan 8199874941, Iran
5Faculty of Chemical Engineering, Naghshe Jahan University, Isfahan 8199874941, Iran
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Abstract - Quercetin has high level antioxidant and
antiradical properties that are consideredbeneficialtocancer
treatment. However, therapeutic applications of quercetin
have been restricted due to its limited solubilityandinstability
in physiological medium. The purpose of this study was to
develop quercetin – Loaded carbon nanotube and to evaluate
the potential of the carrier as a topical delivery system in
cancer therapy. The drug delivery system has been designed
based on functionalized carbon nanotubes by chitosan. Then,
quercetin was conjugated to the carrier. The highest loading
efficiency (38%) was achieved at 4oC and equal initial weight
ratio of drug/carrier. The drug delivery system is stableunder
neutral pH conditions (pH 7.4), while effectively released
quercetin at reduced pH conditions (pH 5.5). In vitro
cytotoxicity assays showed that functionalized carbon
nanotube did not exhibit notable toxicity against Hela cells;
whereas, the cytotoxicity of quercetin conjugated carrier
increased significantly in comparison with pure quercetin.
Hence, such a targeting nanocarrier isasuitablecandidate for
targeted drug delivery in tumor therapy.
Key Words: Carbon nanotube, Quercetin, Chitosan,
targeting, Drug delivery, Cancer therapy
1.INTRODUCTION
Cancer is one of the main causes of death in the worldand its
victims increase every day [1]. Quercetin (3, 3´, 4´, 5´-7-
penta-hydroxy flavone) (fig1) is one of the most abundant
flavonoids in plants found in fruits, vegetablesandherbs [2].
It has a broad extent of chemotherapeutic properties for
many diseases such as anti-cancer, anti-inflammatory, anti-
viral, antiradical and anti-oxidant [3, 4]. However, its
superior anti-cancer activity is well demonstrated in colon,
breast, ovarian and lung cancer cells [5,6]. In vivo reversal
efficacy of quercetin is not satisfactory when it is
administered systemically via free drug due to its low
solubility in aqueous media (7.7 µg/mL in water), poor
permeability, low bioavailability (about 1% in men) [7],
biodegradation limits and high binding ratioofdrug–plasma
protein (99.4%) [8].
One way to overcome these drawbacks is to entrap/adsorb
this molecule into carriers. Since nanoscale drug delivery
carriers offer potential advantages, which include
enhancement of quercetin solubility and bioavailability,
improved tissue macrophages distribution, enhancementof
pharmacological activity, sustained delivery, and protection
from physical and chemical degradation [9,10], a number of
carriers such as liposomes [11,12,13], lipids [14,15,16],
micelles [17, 18], polymeric nanoparticles[19,20],magnetic
nanoparticles [21, 22] and graphene [23] have been applied
for entrapment of quercetin. Recently, single walled carbon
nanotubes (SWNTs) have beenappliedfortargetingdelivery
of various anti-cancer drugs such as doxorubicin and
paclitaxel [1, 24]. Since single walled carbon nanotubes
(SWNT) provide unique properties such ashighsurfacearea
(1300 m2/gr) allowing for higher drug loading and
possibility for accompanying additional therapeutic ligands
through surface functionalization, recently they have
attracted many attentions in the treatment and diagnosis of
cancer.
However, drug delivery systems based on SWNTs still face
critical challenges. They are potentially toxic and extremely
hydrophobic. Functionalization of carbon nanotubes by
covalent and non-covalent interactions can improve their
compatibility and cellular uptake, decrease their
hydrophobicity, and, make them nontoxic. Surfactants [27],
peptides and polymers [1,28] have been used to modify
SWNT via non covalent interactions. Among the frequently
used biological species, chitosanismuchmoreattractive due
to its biodegradability and biocompatibility which can
endow the SWNTs excellent water solubility and good
biological compatibility [29,30]. Moreover, presence of
amine group in chitosan structure can facilitate attachment
of targeting ligands to the drug delivery system [31,32]. In
this paper, for the first time, to the best our knowledge,
single walled carbon nanotube was used to targeting
delivery of quercetin. Chitosan have been applied to
functionalization of carbon nanotubes. First, theinfluence of
the binding conditions during the drug loading step (e.g.,
temperature, initial weight ratio of drug/carrier and initial
amount of chitosanappliedforfunctionalization)onthedrug
International Research Journal of Engineering and Technology (IRJET) e-ISSN: 2395-0056
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loading efficiency was investigated. Then pH dependent
release of the drug was demonstrated which promoted its
release inside tumour cellsuponinternalization.Finally,folic
acid (fig 1b) was also applied to the nanotube surface to
endow targeting ability to the carrier and the therapeutic
efficacy of the drug delivery system was determined by in
vitro cell viability assays.
(a)
HN
HN
CO2H
CO2H
O
HN
N N
N
O
NH2
(b)
Figure 1. Molecular structures of (a) quercetin and (b) folic
acid
2-Experimental procedure
2.1. Materials and Methods
Single walled carbon nanotubes were purchased from
Neutrino Company (Tehran, Iran). (Purity >99%, length
>5µm, diameter 1–2 nm, surface area >380 m2/g). Low
molecular weight chitosan (~50kDa) with 75-85% degreeof
deacetylation,folicacid,ethanol(≥99.5%),N,N′-dicyclohexyl
carbodiimide (DCC), dimethylsulfoxide (DMSO), 3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide
(MTT) and Quercetin (QU) were obtained from Sigma-
Aldrich. Fetal bovine serum (FBS), RPMI 1640 and
penicillin/streptomycin were obtained from Gibco Inc.
Human cervical cancer HeLa cells were purchased from the
Pasteur Institute of Iran, Tehran, Iran. Analytical reagent
grade salts and chemicals were used without further
purification. HPLC grade (> 99%) solvents were used for
these studies.
UV-Visible spectroscopy (UV-2501, Shimadzu, Japan) was
used for quercetin concentration measurement. Fourier
transform infrared (FTIR) (Perkin–Elmer, Spectrum65) and
CHNS elemental analyzer (LECO 932, USA) were used for
characterization of functionalgroups.Infrared(IR)spectraof
the samples were scanned in the range from 400 to4000
cm−1and obtainedat a resolutionof4cm−1withaminimum
of 256 scan per spectrum.
2.2. Functionalization of single walled carbonnanotubes
As raw SWNTs are often too long with bundledoraggregated
structures contaminated by heavy metal impurities, Single
walled carbon nanotubes were cut and purified through
oxidative acid treatment using the proceduredocumentedin
the literature [33]. Briefly, 1g of the SWNTs were suspended
in 250 mL solution of nitric acid (65%) and sulphuric acid
(98%) in a volume ratio of 2:3 then refluxed for24 h at 50 °C.
The resultantmixturewasdilutedwithultra-distillatedwater
followed by centrifugation for several timestoobtainneutral
ph. Finally, the precipitated oxidized SWNTs (ox-SWNT)
containing carboxylicacid groupswerecollectedanddriedat
60°C over night. For functionalization of ox-SWNTs with
chitosan, 50 mg of ox-SWNT and predetermined amount of
chitosan in different chitosan/ ox-SWNTs weight ratio (1/1,
1/2, 1/3, 1/4) were suspended in PBS (pH 7.4, 100ml); and
sonicated for 1h. Then, mixture was stirred at room
temperature for 16 h. The product was filtered and rinsed
with distilled water to remove the unbound polymer; finally,
the solid (CS-SWNT) was collected and freeze dried.
2.3. Folate targeting
Folic acid (FA) was conjugated to the carrier, and make it
targetable to cancer cells. Folic acid (6 mg) was stirredwitha
mixture of 10 mg DDC and 10 ml DMSO for 1 h at room
temperature. Then, CS-SWNTs (3 mg) were added to the
mixture and stirred for 16 h at room temperature in
darkness. The unreacted folic acid was removed by filtration
and washing with distilled water. Finally, the resultant solid
was dried at room temperature and named as FA-CS-SWNT.
2.4. Drug loading
Quercetin loading onto carriers was performed by mixing10
mg of carrier and pre-determined weight of quercetin in
quercetin/CS-SWNTsweightratio(1/1thenbothcomponents
were submerged in 50 ml methanol and stirred for 16 h. The
suspension was centrifuged (13000 rpm, 30 min), filtrated,
and washed thoroughly with methanol until the filtrate
became colorless. Finally, the products (known as QU-ox-
SWNTsandQU-CS-SWNTs)werecollectedandfreezedriedat
20 oC for 24 h. The above-mentioned procedure was
conducted in 4, 25 and 60oC. The amount of unbound
quercetin in the filtrate measured by UV/vis absorption
spectroscopy at 370 nm (the characteristic absorbance of
quercetin in methanol) respective to a calibration curve
accomplished under the same conditions [34]. The
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spectroscopy results were used to calculate drug loading
efficiency according to the following equations:
Drug loading efficiency (%) = 100× (wfeed QU-wfree QU)/
Wfeed QU (1)
Where Wfeed QC and wfree QC are the total amount of
quercetin and the amount of unbound quercetin in the
filtrate, respectively.
2.5. Drug release
1 mg drug loaded SWNT was dispersed in 10 ml of solutions
of PBS and methanol (90:10 v/v) in a screw capped tube and
placed in a shaker incubatorat37oCwithconstanthorizontal
shaking at 100 rpm.Atspecifiedtimeintervals,thenanotubes
were separated from the buffer by ultracentrifugation, and
the whole release medium was replaced with fresh one.
These experiments were carried out on pH 7.4 (normal
condition) and pH 5.5 (tumor condition).
2.6. Cell culture & cell viability experiments
Hela cells were cultured in T25 culture flasks in RPMI-1640
medium supplemented with10% fetal bovine serum and
penicillin/streptomycin (100 units/mL penicillin and 100
µg/mL streptomycin) in an incubator at 37oC in which the
CO2 level was maintained at 5%. In order to measure cell
viability, cultured cells were seeded at a density of 104 cells
per well into 96 well plate and incubated for 24 h, then the
medium was replaced with fresh mediums containing 100
µg/mL CS-SWNTs, 100µg/mL FA-CS-SWNTs, 100µg/mLFA-
QU-CS-SWNTs (based on quercetin concentration) and 100
µg/mL free QU at 37oC. In different time intervals (24, 48,
72h), the in vitro cell cytotoxicity was assessed by the MTT
assay through addition of 10µl solution of MTT reagent
(0.5mg/ml) and incubation for 4 h at 37oC. Then,themedium
was discarded and replaced by 100 µL DMSO to dissolve the
formazan crystals of which the absorbance was calculated
after 1 h of incubation at 570 nm using microplate reader.
The percentage viability of cells was calculatedas the ratioof
absorbance of triplicate readings with control wells.
Results and discussion
Functionalization of single walled carbon nanotubes
Fig 2 displays FTIR spectrums of chitosanandCS-SWNT. The
basic peaks of chitosan areat 3200-3500cm−1(O-Hand N-H
stretch), 2850 cm-1 (C-H stretch), 1596 cm-1 (N-H bend),
1420 (C-H bend), and 1090 cm-1 (C-O-C stretch). The
spectrum of CS-SWNT display all the characteristic
absorption bands of both ox-SWNT and chitosan, indicating
of successful adsorptionofchitosanonthecarbonnanotubes
surface.
Figure 2. FTIR spectroscopy chitosan and CS-SWNT
Drug loading
Loading of quercetin on QC-CS-SWNT confirmed by FTIR
spectroscopy (Fig 3). FTIR spectroscopies of quercetin (Fig
3a) shows broad peaks at 3290 and 1358 cm−1 which are
assigned to stretching vibration and phenolic bending of
hydroxyl (O-H) groups. Peaks at 1662 and 1093 cm−1
assigned to (C=O) stretching and C-O stretching vibrations
from (C-O-C). Peaks at 1512 and 932 cm−1 represent the
aromatic C=C stretching and C-H bending vibration of
aromatic group [7]. The basic characteristic peaks of CS-
SWNT (Fig 3b) are at 3200-3500 cm−1 (O-H and N-H
stretch), 1640 cm-1 (N-H bend), 1152 and 1072cm-1 (C-O-C
stretch). Loading of quercetin on CS-SWNT (Fig 3c) leads to
presence of alkene and aromatic stretching vibration at
1630, 1427 and 1377 cm−1 confirm of quercetin loading on
nanotubes. Moreover,peak at932cm−1inquercetin(Fig 3a)
related to bending vibration of C-H bond in aromatic ring,
shifted to 891 cm−1 in QC-CS-SWNT which is due to the
formation of hydrogen bondsbetween quercetinandcarrier.
Figure 3. Fourier transform infrared (FTIR) spectra of (a)
Quercetin, (b) CS-SWNT and (c) QU-CS-SWNT
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In-vitro quercetin releasing
Fig 4a and 4b display cumulative drug release curves for a
duration of 96 hr. at 37 oC, in PBS pH=5.5 and 7.4,
respectively. The in vitro release kinetics shows initial burst
release followed by a slow and sustained release. This burst
release is normally attributed to the fraction of quercetin
which was adsorbed in the surface of the nanotubes. As
shown in Fig 4 quercetin was released slower from the
nanocarrier at neutral conditions (pH 7.4). There was only
about 32 % of the total drug released for QC-CS-SWNT after
96h While under the pH of tumor tissues (pH 5.5) nearly
61.5% was released. In pH 7.4, the hydrogen bonds between
quercetin and SWNTs were tightly bound; while, by
decreasing the pH, quercetin was quickly released due to
competition of H+ ion with functional groups capable of
forming hydrogen bonding with drug. A higher release rate
of quercetin was achieved for QC-CS-SWNT. As, chitosan
increase hydrophilicity of CNT surfaces, which leads to
weakening of the π–π interactions of QC to the CNTsurfaces,
quercetin can be released easier and faster.
(a)
(b)
Figure 4. Drug release of modified SWNTs at (a) pH=7.4
and (b) pH=5.5
Cell viability studies
The control, CS-SWNT, pure quercetin, quercetinconjugated
nanotubes QU-CS-SWNTs and FA-QU-CS-SWNTs were
incubated individuallywithhumancervical cancerHeLa cells
to study the inhibition of the cell growth rate using MTT
assay. Results are shown in figure 5. As shown in this fig,CS-
SWNT displayed no toxic effect on cells,andcell viabilitywas
94% even after 72 h. This clearly demonstrated that
functionalized carbon nanotubes with chitosan have good
biocompatibility. whereasquercetin-loadedSWNTs(QU-CS-
SWNT) with quercetin concentration of 100 µg/ml caused
significant Hella cancer cell death and the cell viability
decreased to 44% after 72 h which could be higher
compared to pure quercetin (57%) at the same
concentration. The toxicity of the Hela cancer cells might be
attributed to the strong binding between the Hela cells and
quercetin molecules due to the partial release of quercetin
from the nanotubes.
The FA-QU-CS-SWNTs exhibits a cell viability of 37% which
demonstratethetargetingdrugcarrier,FA-QU-CS-SWNT had
stronger cytotoxicity than did the non-targetingone(QU-CS-
SWNT). Folic acid increased cellular uptake and
internalization through receptor-mediated endocytosis
mechanism, which lead killing of specific cancer cells for the
targeting drug carrier [35].
Figure 5. Cell viability of the modified SWNTs: Viability of
HeLa cells treated with FA-CS-SWNTs, QU-CS-SWNTs, QU-
FA-CS-SWNTs, CS-SWNTs and free quercetin for 24, 48
and 72 h.
3. CONCLUSIONS
An efficient drug delivery system based on modified SWNTs
was established. Chitosan and folic acid were applied for
functionalization of single walled carbon nanotubes. Then,
an anticancer drug (quercetin) was loaded on the modified
carrier by π-π interactions and hydrogen bonds. The FT-IR
analysis demonstratedthatquercetinformedintermolecular
hydrogen bonding with carriers. The loading efficiency of
quercetin on CS-SWNT was determined to bearound38%
under best conditions (at 4oC, chitosan/ox-SWNT weight
ratio 1/1 and drug / carrier weight ratio 1/1). This means
that 1 gram of chitosan wrapped carbon nanotubesisable to
deliver 0.38 g of quercetin, which an acceptable loading
capacity compared with many other common nanocarriers.
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The introduced drug delivery systems displayed stability
under physiological conditions (pH = 7.4andtemperatureof
37°C), while simulating the cancer cell condition at pH=5.5,
the quercetin was efficiently released (61.5% after 96hfor
QU-CS-SWNT). Based on the data gathered from in vitro
studies, it can be concluded that, chitosan coating of carbon
nanotubes enhances their biocompatibility and thusmaking
them safe and efficient candidate for targeted delivery
applications. Quercetin delivery by FA-CS-SWNT may be
considered as an effective delivery system for improving
pharmacokinetic and properties of quercetin.
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Formulation and in vitro evaluation of quercetin loaded carbon nanotubes for Cancer Targeting

  • 1. International Research Journal of Engineering and Technology (IRJET) e-ISSN: 2395-0056 Volume: 10 Issue: 06 | Jun 2023 www.irjet.net p-ISSN: 2395-0072 © 2023, IRJET | Impact Factor value: 8.226 | ISO 9001:2008 Certified Journal | Page 962 Formulation and in vitro evaluation of quercetin loaded carbon nanotubes for Cancer Targeting Zahra Alimohammadi1, Asma Zamansani2, Azadeh Abreshteh3, Mehrsa Edalaat4, Zohreh Ebrahimi5 1Faculty of Chemical Engineering, Naghshe Jahan University, Isfahan 8199874941, Iran; 2 Faculty of Chemical Engineering, Naghshe Jahan University, Isfahan 8199874941, Iran 3 Faculty of Chemical Engineering, Naghshe Jahan University, Isfahan 8199874941, Iran 4Faculty of Chemical Engineering, Naghshe Jahan University, Isfahan 8199874941, Iran 5Faculty of Chemical Engineering, Naghshe Jahan University, Isfahan 8199874941, Iran ---------------------------------------------------------------------***--------------------------------------------------------------------- Abstract - Quercetin has high level antioxidant and antiradical properties that are consideredbeneficialtocancer treatment. However, therapeutic applications of quercetin have been restricted due to its limited solubilityandinstability in physiological medium. The purpose of this study was to develop quercetin – Loaded carbon nanotube and to evaluate the potential of the carrier as a topical delivery system in cancer therapy. The drug delivery system has been designed based on functionalized carbon nanotubes by chitosan. Then, quercetin was conjugated to the carrier. The highest loading efficiency (38%) was achieved at 4oC and equal initial weight ratio of drug/carrier. The drug delivery system is stableunder neutral pH conditions (pH 7.4), while effectively released quercetin at reduced pH conditions (pH 5.5). In vitro cytotoxicity assays showed that functionalized carbon nanotube did not exhibit notable toxicity against Hela cells; whereas, the cytotoxicity of quercetin conjugated carrier increased significantly in comparison with pure quercetin. Hence, such a targeting nanocarrier isasuitablecandidate for targeted drug delivery in tumor therapy. Key Words: Carbon nanotube, Quercetin, Chitosan, targeting, Drug delivery, Cancer therapy 1.INTRODUCTION Cancer is one of the main causes of death in the worldand its victims increase every day [1]. Quercetin (3, 3´, 4´, 5´-7- penta-hydroxy flavone) (fig1) is one of the most abundant flavonoids in plants found in fruits, vegetablesandherbs [2]. It has a broad extent of chemotherapeutic properties for many diseases such as anti-cancer, anti-inflammatory, anti- viral, antiradical and anti-oxidant [3, 4]. However, its superior anti-cancer activity is well demonstrated in colon, breast, ovarian and lung cancer cells [5,6]. In vivo reversal efficacy of quercetin is not satisfactory when it is administered systemically via free drug due to its low solubility in aqueous media (7.7 µg/mL in water), poor permeability, low bioavailability (about 1% in men) [7], biodegradation limits and high binding ratioofdrug–plasma protein (99.4%) [8]. One way to overcome these drawbacks is to entrap/adsorb this molecule into carriers. Since nanoscale drug delivery carriers offer potential advantages, which include enhancement of quercetin solubility and bioavailability, improved tissue macrophages distribution, enhancementof pharmacological activity, sustained delivery, and protection from physical and chemical degradation [9,10], a number of carriers such as liposomes [11,12,13], lipids [14,15,16], micelles [17, 18], polymeric nanoparticles[19,20],magnetic nanoparticles [21, 22] and graphene [23] have been applied for entrapment of quercetin. Recently, single walled carbon nanotubes (SWNTs) have beenappliedfortargetingdelivery of various anti-cancer drugs such as doxorubicin and paclitaxel [1, 24]. Since single walled carbon nanotubes (SWNT) provide unique properties such ashighsurfacearea (1300 m2/gr) allowing for higher drug loading and possibility for accompanying additional therapeutic ligands through surface functionalization, recently they have attracted many attentions in the treatment and diagnosis of cancer. However, drug delivery systems based on SWNTs still face critical challenges. They are potentially toxic and extremely hydrophobic. Functionalization of carbon nanotubes by covalent and non-covalent interactions can improve their compatibility and cellular uptake, decrease their hydrophobicity, and, make them nontoxic. Surfactants [27], peptides and polymers [1,28] have been used to modify SWNT via non covalent interactions. Among the frequently used biological species, chitosanismuchmoreattractive due to its biodegradability and biocompatibility which can endow the SWNTs excellent water solubility and good biological compatibility [29,30]. Moreover, presence of amine group in chitosan structure can facilitate attachment of targeting ligands to the drug delivery system [31,32]. In this paper, for the first time, to the best our knowledge, single walled carbon nanotube was used to targeting delivery of quercetin. Chitosan have been applied to functionalization of carbon nanotubes. First, theinfluence of the binding conditions during the drug loading step (e.g., temperature, initial weight ratio of drug/carrier and initial amount of chitosanappliedforfunctionalization)onthedrug
  • 2. International Research Journal of Engineering and Technology (IRJET) e-ISSN: 2395-0056 Volume: 10 Issue: 06 | Jun 2023 www.irjet.net p-ISSN: 2395-0072 © 2023, IRJET | Impact Factor value: 8.226 | ISO 9001:2008 Certified Journal | Page 963 loading efficiency was investigated. Then pH dependent release of the drug was demonstrated which promoted its release inside tumour cellsuponinternalization.Finally,folic acid (fig 1b) was also applied to the nanotube surface to endow targeting ability to the carrier and the therapeutic efficacy of the drug delivery system was determined by in vitro cell viability assays. (a) HN HN CO2H CO2H O HN N N N O NH2 (b) Figure 1. Molecular structures of (a) quercetin and (b) folic acid 2-Experimental procedure 2.1. Materials and Methods Single walled carbon nanotubes were purchased from Neutrino Company (Tehran, Iran). (Purity >99%, length >5µm, diameter 1–2 nm, surface area >380 m2/g). Low molecular weight chitosan (~50kDa) with 75-85% degreeof deacetylation,folicacid,ethanol(≥99.5%),N,N′-dicyclohexyl carbodiimide (DCC), dimethylsulfoxide (DMSO), 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and Quercetin (QU) were obtained from Sigma- Aldrich. Fetal bovine serum (FBS), RPMI 1640 and penicillin/streptomycin were obtained from Gibco Inc. Human cervical cancer HeLa cells were purchased from the Pasteur Institute of Iran, Tehran, Iran. Analytical reagent grade salts and chemicals were used without further purification. HPLC grade (> 99%) solvents were used for these studies. UV-Visible spectroscopy (UV-2501, Shimadzu, Japan) was used for quercetin concentration measurement. Fourier transform infrared (FTIR) (Perkin–Elmer, Spectrum65) and CHNS elemental analyzer (LECO 932, USA) were used for characterization of functionalgroups.Infrared(IR)spectraof the samples were scanned in the range from 400 to4000 cm−1and obtainedat a resolutionof4cm−1withaminimum of 256 scan per spectrum. 2.2. Functionalization of single walled carbonnanotubes As raw SWNTs are often too long with bundledoraggregated structures contaminated by heavy metal impurities, Single walled carbon nanotubes were cut and purified through oxidative acid treatment using the proceduredocumentedin the literature [33]. Briefly, 1g of the SWNTs were suspended in 250 mL solution of nitric acid (65%) and sulphuric acid (98%) in a volume ratio of 2:3 then refluxed for24 h at 50 °C. The resultantmixturewasdilutedwithultra-distillatedwater followed by centrifugation for several timestoobtainneutral ph. Finally, the precipitated oxidized SWNTs (ox-SWNT) containing carboxylicacid groupswerecollectedanddriedat 60°C over night. For functionalization of ox-SWNTs with chitosan, 50 mg of ox-SWNT and predetermined amount of chitosan in different chitosan/ ox-SWNTs weight ratio (1/1, 1/2, 1/3, 1/4) were suspended in PBS (pH 7.4, 100ml); and sonicated for 1h. Then, mixture was stirred at room temperature for 16 h. The product was filtered and rinsed with distilled water to remove the unbound polymer; finally, the solid (CS-SWNT) was collected and freeze dried. 2.3. Folate targeting Folic acid (FA) was conjugated to the carrier, and make it targetable to cancer cells. Folic acid (6 mg) was stirredwitha mixture of 10 mg DDC and 10 ml DMSO for 1 h at room temperature. Then, CS-SWNTs (3 mg) were added to the mixture and stirred for 16 h at room temperature in darkness. The unreacted folic acid was removed by filtration and washing with distilled water. Finally, the resultant solid was dried at room temperature and named as FA-CS-SWNT. 2.4. Drug loading Quercetin loading onto carriers was performed by mixing10 mg of carrier and pre-determined weight of quercetin in quercetin/CS-SWNTsweightratio(1/1thenbothcomponents were submerged in 50 ml methanol and stirred for 16 h. The suspension was centrifuged (13000 rpm, 30 min), filtrated, and washed thoroughly with methanol until the filtrate became colorless. Finally, the products (known as QU-ox- SWNTsandQU-CS-SWNTs)werecollectedandfreezedriedat 20 oC for 24 h. The above-mentioned procedure was conducted in 4, 25 and 60oC. The amount of unbound quercetin in the filtrate measured by UV/vis absorption spectroscopy at 370 nm (the characteristic absorbance of quercetin in methanol) respective to a calibration curve accomplished under the same conditions [34]. The
  • 3. International Research Journal of Engineering and Technology (IRJET) e-ISSN: 2395-0056 Volume: 10 Issue: 06 | Jun 2023 www.irjet.net p-ISSN: 2395-0072 © 2023, IRJET | Impact Factor value: 8.226 | ISO 9001:2008 Certified Journal | Page 964 spectroscopy results were used to calculate drug loading efficiency according to the following equations: Drug loading efficiency (%) = 100× (wfeed QU-wfree QU)/ Wfeed QU (1) Where Wfeed QC and wfree QC are the total amount of quercetin and the amount of unbound quercetin in the filtrate, respectively. 2.5. Drug release 1 mg drug loaded SWNT was dispersed in 10 ml of solutions of PBS and methanol (90:10 v/v) in a screw capped tube and placed in a shaker incubatorat37oCwithconstanthorizontal shaking at 100 rpm.Atspecifiedtimeintervals,thenanotubes were separated from the buffer by ultracentrifugation, and the whole release medium was replaced with fresh one. These experiments were carried out on pH 7.4 (normal condition) and pH 5.5 (tumor condition). 2.6. Cell culture & cell viability experiments Hela cells were cultured in T25 culture flasks in RPMI-1640 medium supplemented with10% fetal bovine serum and penicillin/streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in an incubator at 37oC in which the CO2 level was maintained at 5%. In order to measure cell viability, cultured cells were seeded at a density of 104 cells per well into 96 well plate and incubated for 24 h, then the medium was replaced with fresh mediums containing 100 µg/mL CS-SWNTs, 100µg/mL FA-CS-SWNTs, 100µg/mLFA- QU-CS-SWNTs (based on quercetin concentration) and 100 µg/mL free QU at 37oC. In different time intervals (24, 48, 72h), the in vitro cell cytotoxicity was assessed by the MTT assay through addition of 10µl solution of MTT reagent (0.5mg/ml) and incubation for 4 h at 37oC. Then,themedium was discarded and replaced by 100 µL DMSO to dissolve the formazan crystals of which the absorbance was calculated after 1 h of incubation at 570 nm using microplate reader. The percentage viability of cells was calculatedas the ratioof absorbance of triplicate readings with control wells. Results and discussion Functionalization of single walled carbon nanotubes Fig 2 displays FTIR spectrums of chitosanandCS-SWNT. The basic peaks of chitosan areat 3200-3500cm−1(O-Hand N-H stretch), 2850 cm-1 (C-H stretch), 1596 cm-1 (N-H bend), 1420 (C-H bend), and 1090 cm-1 (C-O-C stretch). The spectrum of CS-SWNT display all the characteristic absorption bands of both ox-SWNT and chitosan, indicating of successful adsorptionofchitosanonthecarbonnanotubes surface. Figure 2. FTIR spectroscopy chitosan and CS-SWNT Drug loading Loading of quercetin on QC-CS-SWNT confirmed by FTIR spectroscopy (Fig 3). FTIR spectroscopies of quercetin (Fig 3a) shows broad peaks at 3290 and 1358 cm−1 which are assigned to stretching vibration and phenolic bending of hydroxyl (O-H) groups. Peaks at 1662 and 1093 cm−1 assigned to (C=O) stretching and C-O stretching vibrations from (C-O-C). Peaks at 1512 and 932 cm−1 represent the aromatic C=C stretching and C-H bending vibration of aromatic group [7]. The basic characteristic peaks of CS- SWNT (Fig 3b) are at 3200-3500 cm−1 (O-H and N-H stretch), 1640 cm-1 (N-H bend), 1152 and 1072cm-1 (C-O-C stretch). Loading of quercetin on CS-SWNT (Fig 3c) leads to presence of alkene and aromatic stretching vibration at 1630, 1427 and 1377 cm−1 confirm of quercetin loading on nanotubes. Moreover,peak at932cm−1inquercetin(Fig 3a) related to bending vibration of C-H bond in aromatic ring, shifted to 891 cm−1 in QC-CS-SWNT which is due to the formation of hydrogen bondsbetween quercetinandcarrier. Figure 3. Fourier transform infrared (FTIR) spectra of (a) Quercetin, (b) CS-SWNT and (c) QU-CS-SWNT
  • 4. International Research Journal of Engineering and Technology (IRJET) e-ISSN: 2395-0056 Volume: 10 Issue: 06 | Jun 2023 www.irjet.net p-ISSN: 2395-0072 © 2023, IRJET | Impact Factor value: 8.226 | ISO 9001:2008 Certified Journal | Page 965 In-vitro quercetin releasing Fig 4a and 4b display cumulative drug release curves for a duration of 96 hr. at 37 oC, in PBS pH=5.5 and 7.4, respectively. The in vitro release kinetics shows initial burst release followed by a slow and sustained release. This burst release is normally attributed to the fraction of quercetin which was adsorbed in the surface of the nanotubes. As shown in Fig 4 quercetin was released slower from the nanocarrier at neutral conditions (pH 7.4). There was only about 32 % of the total drug released for QC-CS-SWNT after 96h While under the pH of tumor tissues (pH 5.5) nearly 61.5% was released. In pH 7.4, the hydrogen bonds between quercetin and SWNTs were tightly bound; while, by decreasing the pH, quercetin was quickly released due to competition of H+ ion with functional groups capable of forming hydrogen bonding with drug. A higher release rate of quercetin was achieved for QC-CS-SWNT. As, chitosan increase hydrophilicity of CNT surfaces, which leads to weakening of the π–π interactions of QC to the CNTsurfaces, quercetin can be released easier and faster. (a) (b) Figure 4. Drug release of modified SWNTs at (a) pH=7.4 and (b) pH=5.5 Cell viability studies The control, CS-SWNT, pure quercetin, quercetinconjugated nanotubes QU-CS-SWNTs and FA-QU-CS-SWNTs were incubated individuallywithhumancervical cancerHeLa cells to study the inhibition of the cell growth rate using MTT assay. Results are shown in figure 5. As shown in this fig,CS- SWNT displayed no toxic effect on cells,andcell viabilitywas 94% even after 72 h. This clearly demonstrated that functionalized carbon nanotubes with chitosan have good biocompatibility. whereasquercetin-loadedSWNTs(QU-CS- SWNT) with quercetin concentration of 100 µg/ml caused significant Hella cancer cell death and the cell viability decreased to 44% after 72 h which could be higher compared to pure quercetin (57%) at the same concentration. The toxicity of the Hela cancer cells might be attributed to the strong binding between the Hela cells and quercetin molecules due to the partial release of quercetin from the nanotubes. The FA-QU-CS-SWNTs exhibits a cell viability of 37% which demonstratethetargetingdrugcarrier,FA-QU-CS-SWNT had stronger cytotoxicity than did the non-targetingone(QU-CS- SWNT). Folic acid increased cellular uptake and internalization through receptor-mediated endocytosis mechanism, which lead killing of specific cancer cells for the targeting drug carrier [35]. Figure 5. Cell viability of the modified SWNTs: Viability of HeLa cells treated with FA-CS-SWNTs, QU-CS-SWNTs, QU- FA-CS-SWNTs, CS-SWNTs and free quercetin for 24, 48 and 72 h. 3. CONCLUSIONS An efficient drug delivery system based on modified SWNTs was established. Chitosan and folic acid were applied for functionalization of single walled carbon nanotubes. Then, an anticancer drug (quercetin) was loaded on the modified carrier by π-π interactions and hydrogen bonds. The FT-IR analysis demonstratedthatquercetinformedintermolecular hydrogen bonding with carriers. The loading efficiency of quercetin on CS-SWNT was determined to bearound38% under best conditions (at 4oC, chitosan/ox-SWNT weight ratio 1/1 and drug / carrier weight ratio 1/1). This means that 1 gram of chitosan wrapped carbon nanotubesisable to deliver 0.38 g of quercetin, which an acceptable loading capacity compared with many other common nanocarriers.
  • 5. International Research Journal of Engineering and Technology (IRJET) e-ISSN: 2395-0056 Volume: 10 Issue: 06 | Jun 2023 www.irjet.net p-ISSN: 2395-0072 © 2023, IRJET | Impact Factor value: 8.226 | ISO 9001:2008 Certified Journal | Page 966 The introduced drug delivery systems displayed stability under physiological conditions (pH = 7.4andtemperatureof 37°C), while simulating the cancer cell condition at pH=5.5, the quercetin was efficiently released (61.5% after 96hfor QU-CS-SWNT). Based on the data gathered from in vitro studies, it can be concluded that, chitosan coating of carbon nanotubes enhances their biocompatibility and thusmaking them safe and efficient candidate for targeted delivery applications. Quercetin delivery by FA-CS-SWNT may be considered as an effective delivery system for improving pharmacokinetic and properties of quercetin. REFERENCES [1] AliMohammadi, Z., Aghamiri,S.F., Zarrabi, A., Talaie, M.R., “A comparative study on non-covalent functionalization of carbon nanotubes bychitosananditsderivativesfordelivery of doxorubicin”, Chemical Physics Letters; vol.642,2015, pp. 22–28. [2] Wu, T.H., Yen, F.L., Lin, L.T., Cham, T.M., "Preparation, physicochemical characterization, and antioxidanteffects of quercetin nanoparticles", Int. J. Pharm., vol 346, 2008, pp. 160–168. [3] Song, X., Zhao, Y., Hou, S., Xu, F. Chen, Q., " Dual agents loaded PLGA nanoparticles: Systematic study of particlesize and drug entrapment efficiency", European. Pharm. Biopharm., vol. 69, 2008, pp. 445–453. [4] Rajesh Kumar, S., Priyatharshni, S., Ponpandian; N, "Quercetin conjugated superparamagnetic magnetite nanoparticles for in-vitro analysis of breast cancer cell lines for chemotherapy applications", J. Colloid and Interface Sci. vol. 436,2014, pp. 234–242. [5] Park, J. H., Saravanakumar, G. and Kim, K. "Targeted delivery of low molecular drugs using chitosan and its derivatives", Adv. Drug Deliv. Rev., 62,2010, pp. 28–41. [6] Wolski,P., Nieszporek, K., and Panczyk. T., “Pegylated and folic acid functionalized carbon nanotubes as pH- controlled carriers of doxorubicin. Molecular dynamics analysis of the stability and drug release mechanism” Phys. Chem. Chem. Phys., vol 19, 2017, pp. 9300-9312. [7] Kumari, A., Yadav, S.K., Pakade, Y.B., Singh, B. and Yadav, S.C., "Development of biodegradable nanoparticles for delivery of quercetin", Colloids Surf., B., 80, 2010, pp.184– 192. [8] Panczyk, T., Wolski,P., Jagusiak, A., and Drach, M.,” Molecular dynamics study of Congo red interaction with carbon nanotubes”, RSC Adv., 2014, 47304-47312. [9] Zarrabi, A., Zarepour, A., Khosravi, A., Alimohammadi, Z., Thakur, V., “Synthesis of Curcumin Loaded Smart pH- Responsive Stealth Liposome as a Novel Nanocarrier for Cancer Treatment” Fibers; vol 9, 2021, 19. [10] AliMohammadi, Z., Aghamiri,S.F., Zarrabi, A., Talaie, M.R., “Liposomal Doxorubicin Delivery Systems: Effects of Formulation and Processing Parameters on Drug Loading and Release Behavior”; Current Drug Delivery. Vol 13. 2016, pp.1065-1070. [11] Z.P. Yuan, L.J. Chen, L.Y. Fan, M.T. Tang, G.L. Yang, H.S. Yang, X.B. Du, G.Q. Wang, W.X. Yao, Q.M. hao, B. Ye, R. Wang, P. Diao, W. Zhang, H.B. Wu, X. Zhao, Y.Q. Wei, “Liposomal Quercetin Efficiently Suppresses Growth of Solid Tumors in Murine Models”, Clin. Cancer Res. 12, 2006, 3193-3199. [12] M.Y. Wong, G.N.C. Chiu, “Liposome formulation of co- encapsulated vincristine and quercetin enhanced antitumor activity in a trastuzumab-insensitive breasttumorxenograft model”, Nanomedicine: Nanotechnology, Biology, and Medicine. 7 ,2011, 834–840. [13] E. Alexopouloua, A. Georgopoulosa, K.A. Kagkadisb, C. Demetzosa,“PreparationandCharacterizationofLyophilized Liposomes with Incorporated Quercetin”, J. Liposome Resh. 16, 2006, 17–25. [14] H. Li, X. Zhao, Y. Ma, G.X. Zhai, L.B. Li, H.X. Lou, “Enhancement of gastrointestinal absorptionof quercetin by solid lipid nanoparticles”, J. Controlled Release.133 ,2009, 238–244. [15] C.Y. Guo, C.F.Yang, Q.L. Li, Q. Tan, Y.W. Xi, W.N. Liu, G.X. Zhai, “Development of a Quercetin-loaded nanostructured lipid carrier for formulation topical delivery”, Int. J. Pharm. 430, 2012, 292-298. [16] S. Scalia, M. Mezzena, “Incorporation of quercetin in lipid microparticles: Effect on photo- andchemical stability”, J Pharm. Biomed. Anal. 49, 2009, 90–94. [17] Q. Tan, W. Liu, G. Guo, G. Zhai, “Preparation and evaluation of quercetin-loaded lecithin-chitosan nanoparticles for topical delivery”, Int. J. Nanomedicine. 6, 2011, 1621–1630. [18] R. Trivedi, U.B. Kompella, “Nanomicellar formulations for sustained drug delivery: strategies and underlying Principles”, Nanomedicine. 5, 2010, 485–505. [19] H.K. Ha, J.K. Kim, M.R. Lee, W.J. Lee, “Formation and characterization of quercetin-loaded chitosan oligosaccharide/β-lactoglobulinnanoparticle”,FoodRes.Int. 52, 2013, 82–90. [20] Y. Zhang, Y. Yang, K. Tang, X. Hu, G. Zou, “Physicochemical Characterization and Antioxidant Activity
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