Cu-In-S/ZnS@carboxymethylcellulose supramolecular structures: Fluorescent nanoarchitectures for targeted-theranostics of cancer cells

Carbohydrate Polymers 247 (2020) 116703

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Cu-In-S/ZnS@carboxymethylcellulose supramolecular structures:
Fluorescent nanoarchitectures for targeted-theranostics of cancer cells

T

Alexandra A.P. Mansura, Josué C. Amaral-Júniora, Sandhra M. Carvalhoa,b, Isadora C. Carvalhoa,
Herman S. Mansura,*
a
Center of Nanoscience, Nanotechnology, and Innovation - CeNano2I, Department of Metallurgical and Materials Engineering, Federal University of Minas Gerais – UFMG,
Av. Antônio Carlos, 6627, Belo Horizonte, MG, Brazil
b
Department of Preventive Veterinary Medicine, Veterinary School, Federal University of Minas Gerais – UFMG, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords:
Polysaccharides
Carboxymethylcellulose
Fluorescent nanomaterials
Quantum dots
Drug delivery
Folate receptor

Cancer targeting
Cell imaging

Although the field of oncology nanomedicine has shown indisputable progress in recent years, cancer remains
one of the most lethal diseases, where the early diagnosis plays a pivotal role in the patient's prognosis and
therapy. Herein, we report for the first time, the synthesis of biocompatible nanostructures composed of Cu-In-S
and Cu-In-S/ZnS nanoparticles functionalized with carboxymethylcellulose biopolymer produced by a green
aqueous process. These inorganic-organic colloidal nanohybrids developed supramolecular architectures stabilized by chemical functional groups of the polysaccharide shell with the fluorescent semiconductor nanocrystal
core, which were extensively characterized by several morphological and spectroscopical techniques. Moreover,
these nanoconjugates were covalently bonded with folic acid via amide bonds and electrostatically conjugated
with the anticancer drug, producing functionalized supramolecular nanostructures. They demonstrated nanotheranostics properties for bioimaging and drug delivery vectorization effective for killing breast cancer cells
in vitro. These hybrids offer a new nanoplatform using fluorescent polysaccharide-drug conjugates for cancer
theranostics applications.

1. Introduction
Oncotherapy has experienced extraordinary progress in recent
decades, although cancer remains a burden as one of the deadliest
diseases of the current century.
Particularly, breast cancer (BC) is presently the utmost prevalent
type of female cancer worldwide (Chen, Zhang, Zhu, Xie, & Chen, 2017;
Mendes, Kluskens, & Rodrigues, 2015; Shi, Kantoff, Wooster, &
Farokhzad, 2017; Sivakumar et al., 2013; Wang, Zhu, Xu, & Wang,
2019; Wang, Zhong et al., 2019) where triple-negative breast cancer
(TNBC), is recognized as an aggressive and metastatic type of BC
(∼15−20 %), posing challenges for oncologists. Additionally, traditional chemotherapy is commonly affected by low cell specificity and
selectivity, severe side-effects, and normally causing drug resistance.
For instance, doxorubicin (DOX) has demonstrated to display high anticancer activity in chemotherapy, including BC, but with limitations
due to the necessity of administration at very high doses to reach the
tumor site. Consequently, DOX repeatedly causes severe side-effects

and body dysfunctions in BC patients (Wang, Zhu et al., 2019; Wang,
Zhong et al., 2019). Nowadays, the effective strategy against cancer
should focus on the earliest possible diagnosis and the specific targeting
therapy towards cancer cells while preserving healthy cells, and minimizing collateral effects (Chen et al., 2017; Shi et al., 2017). Hence,
nanotheranostic comprising diagnosis and therapy integrated into nanostructures has emerged as a new powerful weapon against cancer
(Mansur, Mansur, Soriano, & Lobato, 2014).
In the realm of 'smartly' designed theranostic nanomaterials, the
amalgamation of components from distinct nature, such as inorganic
nanoparticles with organic molecules and drugs, termed as nanohybrids, can offer virtually unlimited possibilities for the diagnosis and
therapy of cancer. The "hard matter" portion of the hybrid nanosystems,
referred to as the core, is usually composed of inorganic nanomaterials
such as metallic nanoparticles (Capanema et al., 2019), superparamagnetic nanoparticles (Carvalho et al., 2019), and semiconductor
quantum dots (Mansur et al., 2014). These nanomaterials often possess
one or more properties, such as magnetic, optical, electronic,

⁎

Corresponding author at: Federal University of Minas Gerais, Av. Antônio Carlos, 6627 – Escola de Engenharia, Bloco 2 – Sala 2233, 31.270-901, Belo Horizonte,
MG, Brazil.
E-mail addresses: (A.A.P. Mansur), (J.C. Amaral-Júnior),
(S.M. Carvalho), (I.C. Carvalho), (H.S. Mansur).
/>Received 1 April 2020; Received in revised form 9 June 2020; Accepted 25 June 2020
Available online 29 June 2020
0144-8617/ © 2020 Elsevier Ltd. All rights reserved.


Carbohydrate Polymers 247 (2020) 116703

A.A.P. Mansur, et al.

QDs, polymers, and drugs for cancer nanotheranostics (Mansur,
Mansur, Soriano, & Lobato, 2014; Mansur et al., 2019). Surprisingly,
this theme is still in the early stages and barely reported (Jiang & Tian,
2018; Mansur et al., 2019). No previous report was found where nanostructures made of Cu-In-S (CIS) and Cu-In-S/ZnS (ZCIS) QDs and
CMC polymer were produced for nanotheranostics applications in
cancer. Thus, in this work, we hypothesize that inorganic-organic nanohybrids composed of fluorescent Cu-In-S/ZnS QDs can be synthesized
by a green aqueous colloidal process using CMC biopolymer simultaneously as capping ligand and targeting macromolecule coupled to folic
acid, and electrostatically complexed with DOX anticancer for producing nanoassemblies. We further hypothesize these nanoassemblies will
perform dual-mode functions, as photoluminescent nanoprobes for
bioimaging, and as polymer-targeted nanocarriers for killing TNBC cells
in vitro relying on a nanotheranostic strategy.

biochemical, etc., which is crucial for performing the detection and
biosensing for the diagnosis of cancer. Analogously, the "soft matter"
portion of the hybrid nanostructures, known as the shell layer, is
usually made by organic components, such as polymers, biomolecules,
and conjugates, which play a pivotal role on the chemical stability of
the systems and as drug carriers, as well as ascribe the biological
functionalization for affinity recognition of the cancerous cells and
tissues (Mansur et al., 2014, 2018). Thus, colloidal semiconductor
quantum dots (QDs) have been one of the most preferred choices of
inorganic core nanomaterials for diagnosis and biosensing applications
due to their unique amalgamation of optoelectronic properties (Jiang &
Tian, 2018; Mansur et al., 2014, 2019). QDs are versatile nanomaterials
encompassing a narrow and strong photoluminescence emission band,
which can be adjusted by the chemical composition and the size of the
nanocrystals. However, the intrinsic cytotoxicity of QDs produced from
heavy metal ions (i.e., Cd, Pb) primarily hinders their application in
nanomedicine (Oh et al., 2016). Hence, nontoxic or less toxic semiconductor QDs (e.g., ZnS, Ag-In-S) have been developed with greener
processes for cancer nanotheranostics (Carvalho et al., 2020; Mansur

et al., 2014, 2018; Mansur et al., 2019).
On the other side, polymers and polymer-derived conjugates have
been progressively chosen as the shell layer for building hybrid nanoassemblies. Moreover, a new area termed 'polymer therapeutics' encompasses designed macromolecular systems associated with active
drugs against cancer and other life-threatening diseases. This approach
has been used for generating drug delivery systems (DDS) based on
supramolecular nanostructures such as polymer-drug conjugates,
polymer–protein and polymer-peptides conjugates, polymer-drug
complexes, and polyplexes utilized as powerful tools for battling cancer
(Capanema et al., 2019; Carvalho et al., 2019, 2020; Mansur et al.,
2018).
The amalgamation of carbohydrate-based polymers (e.g., polysaccharides, hyaluronic acid, chitosan, and cellulose) with anticancer
drugs has been progressively researched. Polysaccharides, which are
inherently biocompatible polymers usually extracted from natural renewable sources, can be functionalized for nanomedicine applications.
Among many choices of semi-processed natural polymers, carboxymethylcellulose (CMC) finds extensive use in biology, nutrition,
medicine, and pharmaceuticals. CMC is a commercially available cellulose derivative, which comprises unique physicochemical and biochemical properties, including a remarkable water solubility in a wide
range of pH (e.g., at physiological conditions). CMC biopolymers possess amphiphilic behavior and reactive chemical groups (e.g., hydroxyl
and carboxylic), which permit their functionalization with biomolecules and interactions with insoluble (or low soluble) hydrophobic
drugs. Therefore, the CMC polymer chain can be chemically modified
by grafting to synthesize conjugates with designed nanostructures.
Furthermore, CMC is nontoxic, which has been granted safety approval
by the United States regulation agency (i.e., Food and Drug
Administration, FDA) for parenteral administration in nutritional, biomedical, and pharmaceutical products. Hence, polymer-drug nanosystems have been developed for passive and active targeting drugs to be
delivered to specific sites while minimizing the adverse side-effects and
with improved dose efficiency (Carvalho et al., 2020; Mansur, Mansur,
Soriano, & Lobato, 2014; Mansur et al., 2018). Regarding active targeting, the polymer-based nanocarriers usually are conjugated with a
directing moiety (e.g., proteins, peptides, and cell-target receptors),
thereby permitting preferential accumulation of the anticancer drug
within selected cancer cells or tissues. Especially, folate receptors are
highly expressed by several malignant tumors (e.g., TNBC), while limited or absent in healthy cells (Gazzano et al., 2018; Hansen et al.,
2015; Kayani, Bordbar, & Firuzic, 2018). Thus, folic acid (FA) has been

associated with drug nanocarriers for active targeting folate receptors
frequently overexpressed by cancer cells (Mendes et al., 2015;
Sivakumar et al., 2013). Therefore, a new generation of hybrid nanostructures has emerged, encompassing the properties of semiconductor

2. Materials and methods
Essential information is described in this section, and all of the
materials and standard procedures are detailed at Electronic
Supplementary Material.
2.1. Carboxymethylcellulose characterization
Sodium carboxymethylcellulose (CMC) with the degree of substitution 1.22, average molar mass 250 kDa, and viscosity 660 cps (2 %
in H2O at 25 °C) was supplied by Sigma-Aldrich (Certificate of Analysis
Sigma-Aldrich, Batch # MKBV4486 V). Moreover, the physicochemical
characterization of CMC was carried out using ultraviolet-visible
(UV–vis, CMC solution 0.4 g L−1, transmission mode, Lambda EZ-210/
Perkin-Elmer), photoluminescence (PL, CMC solution 0.4 g L−1, emission spectra at λexc = 325 nm, FluoroMax-Plus–CP/Horiba Scientific),
Fourier transform infrared (FTIR, attenuated total reflectance, CMC cast
film after concentration, Nicolet 6700/Thermo Fischer), and proton
nuclear magnetic resonance (1H-NMR, 20 mg of CMC dissolved in 700
μL de H2O, 64 scans, Avance™III HD NanoBay 400 MHz/Bruker)
spectroscopy techniques. Also, zeta potential (ZP, n = 10, CMC solution
20 g L−1, ZetaPlus/Brookhaven Instruments) assay was performed. The
acid dissociation constant (pKa) of CMC polymer was calculated according to Aggeryd and Olin (1985).
2.2. Synthesis, functionalization, and characterization nanoconjugates
CIS and ZCIS quantum dots were synthesized via an aqueous process. Under magnetic stirring, 2.0 mL of the indium solution (1 × 10−2
M) and 0.12 mL of copper solution (1 × 10−2 M) were added to 42.0
mL of CMC solution (0.4 g L-1, pH = 7.5 ± 0.2) and stirred for 1 min.
Then, 2.0 mL of sulfide solution precursor (1 × 10−2 M) was injected
into the flask, stirred for 10 min, and heated at 90 ± 5 °C for 5 h. This
suspension was left to cool down to room temperature and dialyzed for
24 h against 3 L of distilled water (pH = 5.5 ± 0.2), which was referred

to as CIS@CMC ([In:Cu:S]=[1:0.06:1]).
Then, the CIS nuclei acted as seeds for the deposition of the ZnS
layer producing ZCIS@CMC. Under magnetic stirring, 1.0 mL of zinc
solution (1 × 10−2 M) was added into 42 mL of the CIS@CMC suspension. After 1 min, 1.0 mL of sulfide solution (1 × 10−2 M) was
injected and thermally treated for 5 h at 90 ± 5 °C. The ZCIS colloidal
suspension was dialyzed for 24 h (pH = 5.5 ± 0.2). The overall composition of ZCIS was [In:Cu:S/Zn:S] = [1:0.06:1/0.5:0.5].
The polymer-folate bioconjugate was produced using ZCIS@CMC
QDs for targeted-bioimaging and drug delivery based on N-Ethyl-N'-[3dimethylaminopropyl]carbodiimide hydrochloride (EDC) crosslinking
reaction and L-Arginine as a spacer. The chemical conjugation of folic
acid to ZCIS-CMC conjugates was conducted in two steps. Initially, the
conjugation of L-Arginine to ZCIS@CMC (ZCIS@CMC_L-Arginine) was
performed using EDC at the ratio of L-Arginine:CMC of about 1.0:2.2
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moieties (COO−), which was maintained with the increase of pH
(10.5). In the acidic medium (pH = 3.5), negatively charged carboxylate groups are protonated, forming carboxylic acid (R-COO− + H+/
R-COOH), decreasing the ZP of the macromolecular system.

(w/w). In the sequence, folic acid (FA) was conjugated to the
ZCIS@CMC_L-Arginine using EDC/N-hydroxysulfosuccinimide sodium
salt. This folate modified quantum dot was identified as ZCIS@CMC-FA,
and the reaction yielded a FA:CMC ratio of 1.0:2.2 (w/w). Then, drug
complexes (ZCIS@CMC-FA-DOX) were obtained by electrostatic interactions between negative carboxylate groups from ZCIS@CMC-FA and
cationic doxorubicin (DOX) at a load degree of DOX:CMC of 1.0:1.0 (w/
w). The loading efficiency was calculated (> 99 %) based on the BeerLambert calibration curve. It is noteworthy that pH = 5.5 ± 0.2 was

attained after dialysis and used during the steps of conjugation with FA
and complexation with DOX, as it was favorable for all of the reactions
involved, and for the stability of drug/nanocarrier systems.
Nanomaterials were extensively characterized by several techniques
for assessing their morphological, structural, and spectroscopic features: ultraviolet-visible and photoluminescence spectroscopy (steadystate, 3D excitation-emission curves, and time-correlated single-photon
count, TCSP), transmission electron microscopy (TEM), atomic force
microscopy (AFM), Fourier-transform infrared spectroscopy, X-ray
photoelectron spectroscopy (XPS), zeta potential, and dynamic light
scattering (DLS). Moreover, in vitro drug release of nanocarrier in
comparison to “free” DOX was performed at pH 7.4 (phosphate-buffered saline, PBS, as acceptor medium) by dialysis method using on
Beer-Lambert Law (Mansur et al., 2019).
MTT protocols (Mansur et al., 2019) were used to evaluate the effect
of FA functionalization on the killing efficiency of nanoconjugates after
incubation with folate-deficient cells (FRα-, HEK 293 T and MCF7) and
cells overexpressing folate (FRα+, TNBC) for different times (6 and 24
h). Nanoconjugates were tested at a final concentration of 2.5 nM of
ZCIS nanoparticles (ZCIS@CMC, ZCIS@CMC-FA, and ZCIS@CMC-FADOX) and 5.0 μM of DOX (ZCIS@CMC-FA-DOX). As references, CMC
solution and free DOX at the final concentrations of 10 mg L−1 and 5.0
μM, respectively, were also tested. Statistical significance was tested
using One-way ANOVA followed by Bonferroni's method (α < 0.05).
Confocal laser scanning microscopy (CLSM) experiments of internalization of nanocarrier (ZCIS@CMC-FA-DOX) and controls (CMC, FA,
DOX, and ZCIS@CMC) were performed based on our published reports
(Mansur et al., 2019). Fluorescence was imaged using DAPI, FITC, and
TRITC filters after exposing cell lines (MCF7 and TNBC) to samples for
30 min.
All specific details and protocols related to the materials and experimental procedures are detailed in the Supplementary Material.

3.2. Characterization of CIS and ZCIS nanoparticles
UV–vis spectroscopy of CIS@CMC (Fig. 2A(a)) nanoparticles presented a featureless and broad absorption curve with band edge extending to 700 nm characteristic of Cu-In-S systems. Similar behavior
has been reported in I-III-VI ternary nanocrystals including in Ag-In-S

and Cu-In-S associated with the nanoparticle size polydispersity and the
presence of intragap states arising from the intrinsic defects within the
material (Leach & Macdonald, 2016).
The bandgap energy for the CIS@CMC nanoparticles (EQD =
2.4 ± 0.1 eV) calculated using “TAUC” equation for direct bandgap
semiconductor (Fig. 2A, inset) was blue-shifted from CuInS2 (Ebulk∼1.5
eV) bulk material due to the formation of ternary nanostructures in the
quantum confinement regime (Kolny-Olesiak & Weller, 2013). Based on
this UV–vis spectroscopy analysis and considering the thermodynamics
aspects associated with the high surface-to-volume ratio of the nanocolloids, these results supported the hypothesis that the ternary CIS
quantum dots were effectively nucleated and stabilized by CMC polysaccharide ligand at room temperature using green colloidal chemistry.
The ZnS layers grown onto CIS@CMC QDs nanocrystals followed by
the thermal annealing (at 95 °C for 5 h) caused a further blue-shift of
absorption spectrum (ZCIS@CMC, Fig. 2A(b)), which was related to
alloying by diffusion of ZnS outlayer with CIS core, increasing the energy bandgap of the material due to the deposition of a wider bandgap
semiconductor (ZnS, Ebulk = 3.61 eV) to the pristine nanoalloys (Leach
& Macdonald, 2016).
Photoluminescence (PL) studies of CIS@CMC and ZCIS@CMC
(Fig. 2B–D) revealed the main features for these core and core-shell
nanocrystals consistent with the literature (Leach & Macdonald, 2016)
irrespective of the ligand used for stabilization. Key findings are summarized as follows: (I) extremely large Stokes shift between PL emission
and absorption curve; (II) broad emission spectra related to sub-gap
transitions and absence of significant band-to-band recombination; (III)
blue-shift of fluorescence after shell growth (∼33 nm at λexc = 325
nm); (IV) very long radiative lifetimes that increase with coating CIS
with a ZnS layer (224 ns for CIS@CMC, 243 ns for ZCIS@CMC); and (V)
drastic increase of quantum yield (QY, ∼300 %) due to the formation of
core-shell nanostructures with semiconductors of type-I band alignment
(from QY = 1.5 % for CIS@CMC and 6.0 % for ZCIS@CMC).
Regarding prospective nanomedicine applications, based on the 3D

excitation-emission spectra (Fig. 2C), both CIS@CMC and ZCIS@CMC
exhibited a wide range of excitation wavelengths (from UV up to 600
nm) associated with a broad defect-based emission window from visible
to NIR. Thus, they behaved as optically active nanosystems suitable for
fluorescent nanomedicine applications.
Moreover, the longer lifetimes (Fig. 2D) observed for these CIS and
ZCIS nanoconjugates compared to typical organic fluorophores (i.e.,
chromogenic dyes) and to other QD nanocrystals (with excitonic
emissions) can enhance the sensitivity (Resch-Genger, Grabolle,
Cavaliere-Jaricot, Nitschke, & Nann, 2008) as well as favors the continuous and long-term tracking by bioimaging in biological processes
(Bailey, Smith, & Nie, 2004).
The TEM images of CIS@CMC (Fig. 3A(a)) endorsed the UV–vis
optical absorption findings showing the formation of monodispersed
ultra-small inorganic cores with mostly spherical morphology and
diameter of 3.7 ± 0.4 nm (PDITEM = 0.011) (Fig. 3A(c)), which is lower
than CuInS2 Bohr radius (2rB ∼4.1 nm) (Kolny-Olesiak & Weller,
2013). The continuous lattice fringes obtained by electron diffraction
patterns image (high-resolution TEM, HRTEM, inset Fig. 2A(a)) evidenced the single-crystalline property CIS@CMC. This feature is important because it revealed the capability of CMC as a hard-base

3. Results and discussion
3.1. Characterization of CMC polymer
Carboxymethylcellulose polysaccharide (CMC) plays a pivotal role
in the nucleation, growth, and stabilization of nanocolloidal dispersions. Thus, in this study, the comprehensive characterization of CMC
was conducted using several spectroscopic analyses and biological assays.
The UV–vis spectroscopy analysis of CMC (Fig. 1A) indicated the
absence of HOMO-LUMO energy transitions in the visible range in
agreement with the optical transparency of CMC solutions (i.e., only UV
electronic transitions). Consequently, the CMC polymer solution did not
show photoluminescent emission (Fig. 1B). The FTIR spectra revealed
the main bands associated with functional groups of CMC (e.g., carboxylic/carboxylates and hydroxyls) in addition to the bands of the

saccharine structure (Fig. 1C and Table S1). In 1H-NMR spectra
(Fig. 1D), resonance signals associated with unsubstituted and substituted hydroxyls were detected (Kono, Oshima, Hashimoto, Shimizu,
& Tajima, 2016). The average pKa = 4.2 ± 0.1 for CMC was calculated,
where the pH-sensitive behavior of the CMC polymer was observed in
the curve of ZP as a function of pH (Fig. 1E). After dissolution of CMC in
water (pH ∼ 7.5 > pKa), the dissociation of Na+ ions rendered a net of
negative charge to the polysaccharide associated with carboxylate
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A.A.P. Mansur, et al.

Fig. 1. Results of characterization of CMC: (A) UV–vis, (B) PL, (C) FTIR, (D) 1H-NMR, and (E) ZP.

carboxylate-rich ligand to decrease the reactivity of In3+ in the
medium. This avoided phase separation during the synthesis due to the
distinct reactivity of In3+ (hard Lewis acid) and Cu+ (soft Lewis acid)
(Leach & Macdonald, 2016). After the formation of the ZnS layer
(ZCIS@CMC, Fig. 3B(b,d)), an increase of the nanoparticle diameter to
4.9 ± 0.7 nm (PDITEM = 0.023) was observed. AFM technique was
selected as a complementary tool to further evaluating the morphology

and size of the CIS@CMC. The 3D AFM image (Fig. 3B(e)) revealed the
nanoparticle immersed in the polymer matrix and confirmed the
spherical morphology of the QD with an estimated size of 15 nm. As
expected, this dimension is relatively larger than the values calculated
for the TEM analyses due to the contributions of CIS inorganic core
surrounded by the polymer shell.

Additionally, XPS analysis was used to investigate the chemical
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Fig. 2. (A) UV–vis (inset: TAUC curve for CIS@CMC), (B) PL spectra (λexc = 325 nm), (C) 3D excitation-emission plots, and (D) Lifetime decay curves of (a)
CIS@CMC and (b) ZCIS@CMC.

species were verified by XPS analysis in CIS QDs due to the absence of
shake-up satellite bands in Cu 2p spectrum in the range of 940−945 eV
(Biesinger, 2017). This result was ascribed to the activity of the CMC
polymer functional groups as reducing agents, where CMC hydroxyl
groups played a pivotal role in the reduction of copper metallic ions
during the aqueous synthesis (Capanema et al., 2019).
Hence, these results proved the hypothesis of the formation of novel
hybrid nanocolloids effectively stabilized in aqueous dispersion by the
carboxymethylcellulose biopolymer, which acted as in situ reducing

composition of these nanoconjugates produced. The XPS spectra confirmed the deposition of ZnS outlayer (ZCIS@CMC, Fig. 3B(f)) based on
the Zn 2p region analysis that presented a doublet at 1044.7 eV (2p1/2)
and 1021.7 eV (2p3/2) associated with Zn-S (Zn2+). Moreover, the XPS
spectra of ternary CIS (Cu-In-S) and quaternary ZCIS (Cu-In-S/ZnS) QDs
showed the chemical elements with their respective oxidation states
(i.e., Cu+, In3+, S2−) (Fig. S1). These ions were detected with the same
oxidation states of the respective salt precursors, except for copper. Cu
(II) ions were used as salt precursor (Cu(NO3)2 nitrate), but Cu(I)
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Fig. 3. (a,b) TEM images, (c,d) Histogram of size distribution, and (e) 3D topographical AFM image, and (f) XPS spectra of Zn 2p region of (A) CIS@CMC (leftcolumn) and (B) ZCIS@CMC (right column).

agent and a capping ligand for the nucleation and growth of fluorescent
core-shell inorganic semiconductor nanostructures composed of CIS/
ZnS (ZCIS).
A more in-depth analysis of the core-shell nanoconjugates was
performed by FTIR spectroscopy to investigate the chemical interactions occurring between the functional groups of the CMC ligand and
the inorganic nanocrystal. The FTIR spectra at the range of 4000−2500
cm−1 showed that before the synthesis (Fig. 4A(a)), a set of H-bonds

with water and intra- and interchains involving hydroxyl groups was
observed. After the synthesis of nanoconjugates (Fig. 4A(b,c)), significant changes in the bands assigned to OH groups/hydrogen bonds
were detected, which were associated with the stabilization of QDs and
the chemical reduction of Cu(II) to Cu(I) (Capanema et al., 2019).
In the spectrum range of 1800−800 cm−1, the bands related to
RCOO− and RCOOH moieties in CMC (Fig. 4B(a)) were observed as
well as the stretching vibrations of alcohols and β1-4 glycoside bond
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suggested that the Na+ in sodium salt CMC (supplied polymer) and Mn
in QDs are coordinated to COO− in a combination of monodentate
(Δν1 = 326 cm−1) and bidentate (Δν2 = 174 cm−1) modes (Sutton,
Silva, & Franks, 2015; Zeleňák, Vargová, & Györyová, 2007).
XPS surface analysis of C 1s and O 1s regions were performed to
further investigate the nanointerface CMC-QD, where the polysaccharide played a relevant role in the nucleation, growth, and stabilization of the colloids. The XPS results of CMC ligand are presented
in Fig. 5A,B revealing the different chemical bonds of carbon (CeC/
CeH, CeOH, OeCeO, and O]CeOR) and oxygen (C]O, and CeO/
CeOH) atoms consistent with the chemical structure of the CMC
polysaccharide. After the synthesis of CIS and ZCIS nanoparticles
(Fig. 5C–F), shifts in the binding energy of the band related to
O]CeOR were detected, which were ascribed to R = Na+ being
partially substituted by In3+, Cu+, and Zn2+ (Wang, Zhu et al., 2019;
Wang, Zhong et al., 2019; Yu et al., 2013). Moreover, the XPS analysis
of C and O atomic concentrations indicated no significant changes (i.e.,
C/O atomic ratio), evidencing that thermal treatments of annealing and
alloying have not promoted the oxidation or degradation of the polysaccharide shell layer. These FTIR and XPS results confirmed the interaction of CMC polymer with the nanoparticle surface that favors the
blocking of surface trap states, which resulted in non-radiative recombination pathways, contributing to the increase of the emission
quantum yield.
Zeta potential (ZP) measurements also confirmed the interactions of
QDs with RCOO− groups at the nanocrystal-CMC interfaces. CMC solution at pH 5.5 ± 0.2 typically possesses ZP∼ −50 mV. After the
synthesis of CIS@CMC (pH∼ 5.5), ZP was −32.4 ± 3.0 mV, and as the
reaction proceeded by growing the ZnS layer, ZP value was
−35.4 ± 4.9 mV. The relative reduction of negative charge of CMC in
water medium after CIS and ZCIS QDs nucleation/growth was related to
the complexation reaction. That means, the anionic COO− groups and
the positive metallic ions formed complexes as mono and bidentate ligands, as previously supported by FTIR and XPS analyses. Furthermore,
these ZP values (< −30 mV) indicated that the nanocrystals were
electrostatically stabilized by CMC capping agent with the carboxylate
functional groups combined with steric hindrance effects (Hunter,

1998; Joseph & Singhvi, 2019). Consequently, these aspects were accounted for avoiding the unrestrained growth or agglomeration of the
inorganic nanoparticles (i.e., thermodynamic stabilization), which is
crucial for achieving the semiconductor quantum confinement regime.
The morphological features of these supramolecular architectures dispersed in the aqueous medium were assessed by DLS analysis.
CIS@CMC and ZCIS@CMC systems were produced with hydrodynamic
diameters (Dh) of 21.1 ± 1.8 nm and 45.2 ± 5.2 nm, respectively. The
Dh is assigned to the sum of contributions from the inorganic QDs
("core") and the CMC ("organic shell") of the nanoconjugates, including
solvent within the colloidal structures. These results indicated a lower
volume of solvation for the CIS@CMC that may be associated with the
type of the trivalent indium chelate complex with chemical groups of
the CMC. When compared with ZCIS@CMC systems, the deposition of
ZnS layer provoked a replacement of In3+ species by divalent Zn2+ at
the outmost QD-polymer interface, which caused the expansion (approximately 100 %) of the polymeric shell around the pristine CIS inorganic core. Fig. 6 depicts a schematic representation of the interactions at interface based on the FTIR, XPS, ZP, and DLS results.
+

Fig. 4. FTIR spectra in the range of (A) 4000–2500 cm−1 and (B) 1800–800
cm−1 for (a) CMC, (b) CIS@CMC, and (c) ZCIS@CMC.

(Capanema et al., 2019). After the process of nucleation/growth of CIS
(Fig. 4B(b)) and ZCIS (Fig. 4B(c)), no relevant change was detected in
the energy (i.e., wavenumber) of the vibrations of COO−/COOH groups
of CMC stabilizing ligand (Fig. 4B(a)). However, changes in the relative
intensity of carboxylate/carboxylic bands were observed. An increase of
the absorbance at 1650 cm−1 of COO− species was detected, and the
formation of the Mn+-COO− complex was identified by the decrease of
the peaks associated with RCOOH groups (1730 and 1246 cm−1), as the
Mn+ competes with H+ for complexation. Moreover, the change in the
shape of FTIR spectra between C3eOH and C6eOH stretching bands
and the blue-shift of glycoside peak indicated that they are involved in

the coordination with metal ions/stabilization of QDs, probably due to
the formation of dative bonds between oxygen lone pair electrons and
positive Mn+ (Shukur, Ithnin, & Kadir, 2014). Additionally, the interactions between COO− functional groups of CMC polysaccharide with
QD surfaces were evaluated. In the spectra of CMC, CIS, and ZCIS,
carboxylate groups gave rise to double bands of symmetric (1418 and
1324 cm−1) and asymmetric (1650 and 1592 cm−1) stretching indicating the existence of two different modes of binding to metallic
ions. The type of coordination may be evaluated from the wavenumber
differences between the asymmetric and symmetric vibration (Δν1 and
Δν2). Based on the literature, Δν values obtained from the spectra

3.3. Cancer nanotheranostic applications of core-shell nanohybrids
For targeted-bioimaging and anticancer drug delivery, the conjugation of folic acid to CMC biopolymer was performed in two stages:
(I) coupling L-Arginine to ZCIS@CMC nanostructures; and (II) conjugation of FA membrane receptor to the L-Arginine previously coupled
to ZCIS@CMC forming de ZCIS@CMC-FA nanoconjugates. In both
steps, the EDC "zero-length" crosslinker covalently bonded the amine
groups to the carboxylic/carboxylate species through amide bonds. The
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A.A.P. Mansur, et al.

Fig. 5. XPS analysis of C 1s and O 1s regions acquired for (A,B) CMC (reference), (C,D) CIS@CMC, and (E,F) ZCIS@CMC.

(1592 cm−1) and symmetric (1418 cm−1 and 1324 cm−1) stretching
were observed for all of the samples (Fig. 7A(a–c)). After EDC-mediated
reaction of carboxylates of CMC with N-terminal groups of L-Arginine
(Fig. 7A(b)), the peaks of Amide I at 1640 cm−1 (υ C]O), Amide II at
1540 cm−1 (δ NH and υ CN), and Amide III at 1240 cm−1 (υ CN) were

steps related to the formation of the FA-modified polymer were evaluated using FTIR spectroscopy (from 1800 to 1150 cm−1) for assessing
the main peaks related to the EDC-crosslinking reaction (Fig. 7A). As
references, spectra of L-Arginine (Fig. S2) and FA (Fig. S3) were presented. Bands of COO− groups of CMC associated with asymmetric
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A.A.P. Mansur, et al.

Fig. 6. Schematic representation of (A) Cu-In-S (CIS) and (B) CIS/ZnS (ZCIS) QDs stabilized with polymer CMC. (C) Detail of nanointerface inorganic core-CMC (not
to scale).

acid subpart of FA molecule with side-chain guanidino groups from LArginine, as the amino groups partially reacted at the first stage of the
conjugation process (Psarra et al., 2017).
Fig. 7B presented the fluorescent imaging features of pure DOX drug
(a), pure FA (b), ZCIS@CMC (c), and ZCIS@CMC-FA-DOX complex (d),
at the excitation wavelength of λexc = 375 nm. DOX emission showed

observed (Carvalho et al., 2019). Also, guanidino peaks of L-Arginine at
1675 cm−1 and 1633 cm−1, and the bands at 1475 cm−1 and 1455
cm−1 associated with the eCH2 groups of the aliphatic side chain were
detected. After the second stage Fig. 7A(c), a relative increase of the
intensity of the bands of amides (–CONH-) was identified at 1540 cm−1
and 1240 cm−1 confirming the reaction of carboxylates from glutamic
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A.A.P. Mansur, et al.

Fig. 7. (A) FTIR spectra: (a) ZCIS@CMC, (b) ZCIS@CMC_L-Arginine, and (c) ZCIS@CMC-FA within the range of 1800–1150 cm−1. (B) PL spectra of (a) DOX, (b) FA,
(c) ZCIS@CMC, and (d) ZCIS@CMC-FA-DOX nanocomplexes. (C) Schematic representation of ZCIS@CMC-FA-DOX.

overall balance of the spectral overlapping of fluorescence/emission
curves and molecular interactions intra- and intermolecular species.
Also, they demonstrated the binding of FA and DOX to CMC polymer,
producing hybrid supramolecular colloids with vesicle-like nanostructures.
The acellular in vitro drug release experiment of the ZCIS@CMC-FADOX complexes in comparison to DOX in free form was performed by
the dialysis method (PBS, pH = 7.4 ± 0.2) for 24 h to simulate physiological conditions and the pH of cell culture medium (Fig. S4). The
initial steep rise observed in both systems indicated a burst release
followed by a sustained-release with similar profiles and % of accumulated drug achieved after 24 h. This indicated that the release was

its yellow-red fluorescence signature with a maximum at 592 nm associated with of quinonoid structure (Angeloni, Smulevich, &
Marzocchi, 1982). For FA, the presence of subunits, pterin and 4-aminobenzoyl aromatic rings, rendered a violet-green emission centered at
447 nm (Thomas et al., 2002), and ZCIS@CMC emission spectrum was
previously discussed (Section 3.2). After conjugation with FA and
complexation with DOX, the ZCIS@CMC-FA-DOX nanoassemblies preserved the DOX and FA fluorescence emissions, with a red-shift (5 nm)
for DOX emission and a blue-shift (17 nm) for FA emission, and with a
relative quenching of emission profiles. Conversely, ZCIS@CMC QD
fluorescence was significantly quenched ("Off"). The wavelength shifts
and reduction in PL intensity of emissions resulted from the intricate
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was verified (Fig. S5). These results are of pivotal importance as they
proved the hypothesis of the effect of this novel nanohybrid complex on
active-targeting cancer cells overexpressing folate receptors and drugresistant while simultaneously promoting a relative "protective effect"
towards healthy cells and therefore, minimizing side-effects.
CLSM was used to monitor the trend of cellular uptake of
ZCIS@CMC-FA-DOX nanoconjugates, comparing the images of TNBC
(FRα+) and MCF7 (FRα-) cells incubated with the nanoconjugates (30
min). Reference CLSM images of control samples (cell internalization of
pure CMC, ZCIS@CMC, FA, and DOX, Fig. S6) were obtained. It was
noted the shift of the fluorescence emission of ZCIS@CMC to a shorter
wavelength at intracellular medium (i.e., from green or yellow-red to
blue). Reports have associated this behavior with photooxidation
(Zhang et al., 2006) and the changing of optical properties of QDs by
protein corona (Song et al., 2020).
First, for FITC filter images, a remarkable difference was detected.
For the cell overexpressing folate receptor (Fig. 9A(b)), the green
fluorescence of FA molecules was predominantly observed at the cell
membrane with minor emission at the cytosol. In contrast, for FRαcells (Fig. 9B(b)), the green fluorescence was scattered in the cytoplasm. These differences in the distribution of green signals demonstrated that FA molecules retained targeting activity towards cell
membrane receptors after EDC-mediated conjugation to the ZCIS@CMC
system, vital for the nanotheranostic applications. This was interpreted
as the glutamate groups of FA formed the covalent bonds with
ZCIS@CMC structures, leaving the pteroate sub-part of FA molecule
available to bind to the folate receptors (Chen et al., 2013). Moreover,
these images endorsed the reduction of cell viability observed for TNBC
cells associated with folate receptor-mediated endocytosis of the DOXloaded ZCIS@CMC-FA nanocomplexes.
TRITC filter images depicted the information of anticancer drug
distribution inside the cells. For TNBC cells, yellow-red fluorescence of
DOX (Fig. 9A(c)) was observed overlapping the FA green fluorescence
at the cellular membrane and concentrated at the nucleus. For this cell

line, when the ZCIS@CMC-FA-DOX nanoassembly binds to TNBC folate
receptor, both FA and DOX emitted, but ZCIS emission is quenched
(Fig. 7B). After endocytosis, it was internalized by endosomes for intracellular trafficking along the endosomal-lysosomal pathways releasing electrostatically attached drug cargo (DOX) and FA molecules
coupled to ZCIS@CMC conjugates. The release of these moieties is kinetically favored by the cleavage of amide bonds mediated by the acidic
enzymatic microenvironment of lysosome and the higher solubility of
DOX at lower pHs. After releasing, the DOX molecules migrated and
concentrated in the nucleus causing cell death by intercalation with
DNA and disruption of cell metabolism (Mansur et al., 2018). Also, the
cleavage of amide bonds increased the relative distance between ZCIS
QDs and FA molecules, constraining the contact quenching processes
and, therefore, causing the appearance of ZCIS blue fluorescence as
detected in cytoplasm in DAPI images (Fig. 9A(a)) (Carvalho et al.,
2020). For FRα- cells, FA-mediated endocytosis was not significant,
although passive endocytosis was present. Nonetheless, the same endosomal-lysosomal processes occurred, and DOX fluorescence (TRITC,
Fig. 9B(c)) was localized at the nucleus, with minor intensity at cytosol
where emission of ZCIS was also detected (DAPI, Fig. 9B(a)). It should
be noted that, as it is reported in the literature (Mohan & Rapoport,
2010), the comparison of the red emission localized at the nucleus
between cells cannot be directly correlated to the amount of internalized DOX because the drug fluorescence is dramatically quenched
after intercalation with DNA.
To summarize these results, Fig. 10 depicts a schematically simplified representation of the multifunctional properties for bioimaging and
drug delivery vectorization of fluorescent ZCIS@CMC-FA-DOX nanoarchitecture for targeted-theranostics of cancer cells overexpressing
folate receptors.

Fig. 8. Cell viability results of targeted drug delivery using DOX for HEK 293T,
MCF7 (FRα-), and TNBC (FRα+) cells after incubation for 24 h in comparison
to controls (n = 6; error bars = standard deviation).

dominated by the solubility of the drug in the acceptor medium due to
the low solubility of DOX near to the pKa (∼8.2). However, as expected, at the first 30 min, the release of unbound drug was relatively

higher (> 50 %) than the conjugated DOX due to the modulation
caused by the polymeric nanoarchitecture, which depends on the release of the drug from the nanoconjugates into the dialysis chamber
before the diffusion occurs across the membrane.
The effect of targeting α-folate membrane-receptors was assessed by
MTT assay based on selected cell lines incubated with the samples before and after complexation with DOX. TNBC cancer cell line was selected because of its overexpression of α-folate receptors (FRα+) in the
cellular membrane (Gazzano et al., 2018). Healthy cells (HEK 293T)
and breast cancer cells (MCF7) were used for comparison as folatedeficient (FRα-) cells (Hansen et al., 2015; Kayani et al., 2018).
The cell viability results towards all of the cell lines (Fig. 8, 24 h and
Fig. S5, 6 h) indicated that, after incubation with CMC polymer and
ZCIS@CMC and ZCIS@CMC-FA nanoparticles, there was no statistical
difference compared to the negative control. These findings validated
the hypothesis of the non-cytotoxic behavior of Cu-In-S/ZnS nanoconjugates, which can be potentially applied as fluorescent nanoprobes
for bioimaging cancer cells. Conversely, as expected, the "free DOX"
anticancer drug provoked the death of all cell lines, with high reduction
of cell viability responses towards normal (HEK 293T, 42 %, 24 h) and
cancer cells (MCF7 ∼ 50 % and TNBC ∼ 55 %, 24 h). These results are
consistent with the reported drug resistance of TNBC to conventional
chemotherapy with antitumor agents (Mendes et al., 2015).
After the complexation of DOX with the nanoconjugates forming the
prodrug (ZCIS@CMC-FA-DOX), these nanosystems maintained the
killing activity of the chemotherapeutic agent. However, an increase in
the cell viability response (i.e., lower toxicity) for FRα-deficient cells
was observed (HEK 293T, ∼57 %, Δ = 35 %; MCF7, ∼60 %, Δ = 20
%) compared to "free DOX", which was ascribed to the relevant modulation of DOX release to cells promoted by the polymer-based nanoconjugates as the internalization of DOX loaded in the nanoconjugates
occurs mostly by endocytosis in comparison to the unbound drug that
may enter in cells also by diffusion (Mansur et al., 2018). Conversely,
for FRα + TNBC cell line, the cell lethality response was significantly
increased by approximately 30 % in comparison to the unbound drug as
receptor-mediated endocytosis (RME) allows for a more rapid means of
ligand targeted internalization compared to that of untargeted systems

(Bareford & Swaan, 2007). A more significant behavior was observed
for TNBC cells after 6 h of incubation with nanocarriers, where an
augment of 50 % in killing activity in comparison to the unbound drug
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Fig. 9. CLSM images ((a) DAPI; (b) FITC; (c) TRITC; and (d) channels merged) of cell uptake of ZCIS@CMC-FA-DOX nanoconjugates after incubation for 30 min with:
(A) TNBC (FRα+) and (B) MCF7 cells (FRα-) (scale bar = 10 μm).

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Fig. 10. Illustration of the targeted drug-delivery process of fluorescent ZCIS@CMC-FA-DOX nanoarchitecture (not to scale).

4. Conclusions

CRediT authorship contribution statement

We have successfully demonstrated that novel inorganic-organic
nanohybrids were synthesized by an eco-friendly aqueous process,
using carboxymethylcellulose as capping ligand to produce stable colloidal dispersions of CIS and ZCIS QDs. These nanohybrids showed
ultra-small semiconductor inorganic cores of Cu-In-S (CIS, 2r = 3.7

nm,) and Cu-In-S/ZnS (ZCIS, 2r = 4.9 nm) stabilized by the organic
polymer-based layer of CMC. They formed core-shell supramolecular
colloidal nanostructures, negatively charged (ZP ∼ −35 mV) in aqueous medium at mild pH conditions, with average Dh typically ranging
from 21 nm (CIS) to 45 nm (ZCIS). The cell viability results confirmed
the hypothesis that the nanoconjugates were nontoxic using MTT assay
in vitro. These nanostructures were effectively functionalized with folic
acid biomolecules via the formation of amide bonds with CMC and
complexed with anticancer drug for bioimaging and active targeting
cancer cells. Importantly, the design and development of these surfacefunctionalized fluorescent nanoprobes were successfully proved by
bioimaging and targeted-delivery of the chemotherapeutic drug by the
internalization process and killing of cancer cells in vitro. Thus, based
on the strategy demonstrated in this research, we anticipate that these
new inorganic-organic hybrid supramolecular nanostructures using
polysaccharide-drug bioconjugates can be easily adapted for a broad
range of nanotheranostic applications to work as active fluorescent
multifunctional vehicles for bioimaging, targeting, and killing cancer
cells.

Alexandra A.P. Mansur: Conceptualization, Methodology,
Visualization, Investigation, Formal analysis, Writing - original draft,
Writing - review & editing. Josué C. Amaral-Júnior: Methodology,
Visualization, Investigation, Formal analysis, Writing - review &
editing. Sandhra M. Carvalho: Methodology, Visualization,
Investigation, Validation, Formal analysis, Writing - review & editing.
Isadora C. Carvalho: Methodology, Visualization, Investigation,
Formal analysis, Writing - review & editing. Herman S. Mansur:
Supervision,
Conceptualization,
Methodology,
Visualization,

Validation, Funding acquisition, Writing - original draft, Writing - review & editing, Resources, Project administration.
Declaration of Competing Interest
The authors confirm no competing interests to declare regarding the
publication of this article.
Acknowledgments
The authors would like to thank the staff of the Center of
Nanoscience, Nanotechnology and Innovation-CeNano2I/CEMUCASI/
UFMG for spectroscopy analyses. Also, they express their gratitude to
the staff at the Microscopy Center/UFMG for performing the TEM-EDX
analysis.
Appendix A. Supplementary data

Funding sources
Supplementary material related to this article can be found, in the
online version, at doi: />
The financial support of this study was provided by the Brazilian
Funding Research Agencies: Fundaỗóo de Amparo Pesquisa do Estado
de Minas Gerais FAPEMIG (Grants: UNIVERSAL-APQ-00291-18;
PROBIC-2018; PPM-00760-16); Coordenaỗóo de Aperfeiỗoamento de
Pessoal de Nível Superior - CAPES (Grants: PROINFRA2010–2014;
PROEX-433/2010; PNPD-2014-2019); Financiadora de Estudos e
Projetos - FINEP (Grants: CTINFRA/PROINFRA 2008/2010/2011/
2018); Conselho Nacional de Desenvolvimento Científico e Tecnológico
– CNPq (Grants: PQ1A-303893/2018-4; PQ1B-306306/2014-0; PIBIC2017-18; UNIVERSAL-457537/2014-0; 421312/2018-1).

References
Aggeryd, I., & Olin, A. (1985). Determination of the degree of substitution of sodium
carboxymethylcellulose by potentiometric titration and use of the extended
Henderson-Hasselbalch equation and the simplex method for the evaluation. Talanta,
32, 645–649.

Angeloni, L., Smulevich, G., & Marzocchi, M. P. (1982). Absorption, fluorescence and
resonance Raman spectra of adriamycin and its complex with DNA. Spectrochimica
Acta Part A, 38, 213–217.

13


Carbohydrate Polymers 247 (2020) 116703

A.A.P. Mansur, et al.

peptides for cancer chemotherapy. Bioconjugate Chemistry, 29, 1973–2000.
Mansur, A. A. P., Caires, A. J., Carvalho, S. M., Capanema, N. S. V., Carvalho, I. C., &
Mansur, H. S. (2019). Dual-functional supramolecular nanohybrids of quantum dot/
biopolymer/chemotherapeutic drug for bioimaging and killing brain cancer cells in
vitro. Colloids and Surfaces B, 184, Article 110507.
Mendes, T. F. S., Kluskens, L. D., & Rodrigues, L. R. (2015). Triple negative breast cancer:
Nanosolutions for a big challenge. Advanced Science, 2, Article 1500053.
Mohan, P., & Rapoport, N. (2010). Doxorubicin as a molecular nanotheranostic agent:
Effect of doxorubicin encapsulation in Micelles or nanoemulsions on the ultrasoundmediated intracellular delivery and nuclear trafficking. Molecular Pharmaceutics, 7,
1959–1973.
Oh, E., Liu, R., Nel, A., Gemill, K. B., Bilal, M., Cohen, Y., et al. (2016). Meta-analysis of
cellular toxicity for cadmium-containing quantum dots. Nature Nanotechnology, 11,
479–486.
Psarra, E., König, U., Müller, M., Bittrich, E., Eichhorn, K.-J., Welzel, P. B., et al. (2017). In
situ monitoring of linear RGD-peptide bioconjugation with nanoscale polymer brushes. ACS Omega, 2, 946–958.
Resch-Genger, U., Grabolle, M., Cavaliere-Jaricot, S., Nitschke, R., & Nann, T. (2008).
Quantum dots versus organic dyes as fluorescent labels. Nature Methods, 5, 763–775.
Shi, J., Kantoff, P. W., Wooster, R., & Farokhzad, O. C. (2017). Cancer nanomedicine:
Progress, challenges and opportunities. Nature Reviews Cancer, 17, 20–37.

Shukur, M. F., Ithnin, R., & Kadir, M. F. Z. (2014). Electrical properties of proton conducting solid biopolymer electrolytes based on starch–chitosan blend. Ionics, 20,
977–999.
Sivakumar, B., Aswathy, R. G., Nagaoka, Y., Suzuki, M., Fukuda, T., Yoshida, Y., et al.
(2013). Multifunctional carboxymethyl cellulose-based magnetic nanovector as a
theragnostic system for folate receptor targeted chemotherapy, imaging, and hyperthermia against cancer. Langmuir, 29, 3453–3466.
Song, Y., Wang, H., Zhang, L., Lai, B., Liu, K., & Tan, M. (2020). Protein corona formation
of human serum albumin with carbon quantum dots from roast salmon. Food &
Function, 11, 2358–2367.
Sutton, C. C. R., Silva, G., & Franks, G. V. (2015). Modeling the IR spectra of aqueous
metal carboxylate complexes: Correlation between bonding geometry and stretching
mode wavenumber shifts. Chemistry: A European Journal, 21, 6801–6805.
Thomas, A. H., Lorente, C., Capparelli, A. L., Pokhrel, M. R., Braun, A. M., & Oliveros, E.
(2002). Fluorescence of pterin, 6-formylpterin, 6-carboxypterin and folic acid in
aqueous solution: pH effects. Photochemical & Photobiological Sciences, 1, 421–426.
Wang, Q., Zhong, Y., Liu, W., Wang, Z., Gu, L., Li, X., et al. (2019). Enhanced chemotherapeutic efficacy of the low-dose doxorubicin in breast cancer via nanoparticle
delivery system crosslinked hyaluronic acid. Drug Delivery, 26, 12–22.
Wang, F., Zhu, Y., Xu, H., & Wang, A. (2019). Preparation of carboxymethyl cellulosebased macroporous adsorbent by eco-friendly pickering-MIPEs template for fast removal of Pb2+ and Cd2+. Frontiers in Chemistry, 7, 603.
Yu, X., Tong, S., Ge, M., Wu, L., Zuo, J., Cao, C., et al. (2013). Adsorption of heavy metal
ions from aqueous solution by carboxylated cellulose nanocrystals. Journal of
Environmental Science, 25, 933–943.
Zeleňák, V., Vargová, Z., & Györyová, K. (2007). Correlation of infrared spectra of zinc
(II) carboxylates with their structures. Spectrochimica Acta Part A, 66, 262–272.
Zhang, Y., He, J., Wang, P.-N., Chen, J.-Y., Lu, Z.-J., Lu, D.-R., et al. (2006). Time-dependent photoluminescence blue shift of the quantum dots in living cells: Effect of
oxidation by singlet oxygen. Journal of the American Chemical Society, 128,
13396–13401.

Bailey, R. E., Smith, A. M., & Nie, S. (2004). Quantum dots in biology and medicine.
Physica E, 25, 1–12.
Bareford, L. M., & Swaan, P. W. (2007). Endocytic mechanisms for targeted drug delivery.
Advanced Drug Delivery Reviews, 59, 748–758.

Biesinger, M. C. (2017). Advanced analysis of copper X-ray photoelectron spectra. Surface
and Interface Analysis, 49, 1325–1334.
Capanema, N. S. V., Carvalho, I. C., Mansur, A. A. P., Carvalho, S. M., Lage, A. P., &
Mansur, H. S. (2019). Hybrid hydrogel composed of carboxymethylcellulose–silver
nanoparticles–doxorubicin for anticancer and antibacterial therapies against melanoma skin cancer cells. ACS Applied Nano Materials, 2, 7393–7408.
Carvalho, S. M., Leonel, A. G., Mansur, A. A. P., Carvalho, I. C., Krambrock, K., & Mansur,
H. S. (2019). Bifunctional magnetopolymersomes of iron oxide nanoparticles and
carboxymethylcellulose conjugated with doxorubicin for hyperthermo-chemotherapy
of brain cancer cells. Biomaterials Science, 7, 2102–2122.
Carvalho, I. C., Mansur, H. S., Mansur, A. A. P., Carvalho, S. M., Oliveira, L. C. A., & Leite,
M. F. (2020). Luminescent switch of polysaccharide-peptide-quantum dot nanostructures for targeted-intracellular imaging of glioblastoma cells. Journal of Molecular
Liquids, 304, Article 112759.
Chen, C., Ke, J., Zhou, X. E., Yi, W., Brunzell, J. S., Li, J., et al. (2013). Structural basis for
molecular recognition of folic acid by folate receptors. Nature, 500, 486–489.
Chen, H., Zhang, W., Zhu, G., Xie, J., & Chen, X. (2017). Rethinking cancer nanotheranostics. Nature Reviews Materials, 2, 17024.
Gazzano, E., Rolando, B., Chegaev, K., Salaroglio, I. C., Kopecka, J., Pedrini, I., et al.
(2018). Folate-targeted liposomal nitrooxy-doxorubicin: An effective tool against
pglycoprotein-positive and folate receptor-positive tumors. Journal of Controlled
Release, 270, 37–52.
Hansen, M. F., Greibe, E., Skovbjerg, S., Rohde, S., Kristensen, A. C. M., Jensen, T. R.,
et al. (2015). Folic acid mediates activation of the pro-oncogene STAT3 via the folate
receptor alpha. Cellular Signalling, 27, 1356–1368.
Hunter, R. J. (1998). Zeta potential in colloid science: Principles and applications. Academic
Press.
Jiang, Y., & Tian, B. (2018). Inorganic semiconductor biointerfaces. Nature Reviews
Materials, 3, 473–490.
Joseph, E., & Singhvi, G. (2019). Multifunctional nanocrystals for cancer therapy: A potential nanocarrier. In A. M. Grumezescu (Ed.). Nanomaterials for drug delivery and
therapy (pp. 91–116). Elsevier Inc.
Kayani, Z., Bordbar, A.-K., & Firuzic, O. (2018). Novel folic acid-conjugated doxorubicin
loaded B-Lactoglobulin nanoparticles induce apoptosis in breast cancer cells.

Biomedecine & Pharmacotherapy, 107, 945–956.
Kolny-Olesiak, J., & Weller, H. (2013). Synthesis and application of colloidal CuInS2
semiconductor nanocrystals. ACS Applied Materials & Interfaces, 5, 12221–12237.
Kono, H., Oshima, K., Hashimoto, H., Shimizu, Y., & Tajima, K. (2016). NMR characterization of sodium carboxymethyl cellulose: Substituent distribution and mole
fraction of monomers in the polymer chains. Carbohydrate Polymers, 146, 1–9.
Leach, A. D. P., & Macdonald, J. E. (2016). Optoelectronic properties of CuInS2 nanocrystals and their origin. The Journal of Physical Chemical Letters, 7, 572–583.
Mansur, A. A. P., Mansur, H. S., Soriano, A., & Lobato, Z. I. P. (2014). Fluorescent nanohybrids based on quantum dot−chitosan−antibody as potential cancer biomarkers. ACS Applied Materials & Interfaces, 6, 11403–11412.
Mansur, A. A. P., Carvalho, S. M., Lobato, Z. I. P., Leite, M. F., Cunha, A. S., & Mansur, H.
S. (2018). Design and development of polysaccharide-doxorubicin-Peptide bioconjugates for dual synergistic effects of integrin-targeted and cell-penetrating

14