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

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.

Contents lists available atScienceDirect Carbohydrate Polymers journal homepage:www.elsevier.com/locate/carbpol

Cu-In-S/ZnS@carboxymethylcellulose supramolecular structures:

Fluorescent nanoarchitectures for targeted-theranostics of cancer cells

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 - CeNano 2 I, 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 L E I N F O

Keywords:

Polysaccharides

Carboxymethylcellulose

Fluorescent nanomaterials

Quantum dots

Drug delivery

Folate receptor

Cancer targeting

Cell imaging

A B S T R A C T Although thefield 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 thefirst 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 stabi-lized by chemical functional groups of the polysaccharide shell with thefluorescent 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 na-notheranostics 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,

tradi-tional 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

an-ticancer 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 mini-mizing collateral effects (Chen et al., 2017;Shi et al., 2017) Hence, nanotheranostic comprising diagnosis and therapy integrated into na-nostructures 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 nanohy-brids, 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), super-paramagnetic 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,

https://doi.org/10.1016/j.carbpol.2020.116703

Received 1 April 2020; Received in revised form 9 June 2020; Accepted 25 June 2020

⁎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:alexandramansur.ufmg@gmail.com(A.A.P Mansur),josueamaraljr@gmail.com(J.C Amaral-Júnior),

sandhra.carvalho@gmail.com(S.M Carvalho),isadora.cota@gmail.com(I.C Carvalho),hmansur@demet.ufmg.br(H.S Mansur)

Carbohydrate Polymers 247 (2020) 116703

Available online 29 June 2020

0144-8617/ © 2020 Elsevier Ltd All rights reserved

T

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

semi-conductor 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

na-noassemblies Moreover, a new area termed 'polymer therapeutics'

en-compasses 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.,

poly-saccharides, hyaluronic acid, chitosan, and cellulose) with anticancer

drugs has been progressively researched Polysaccharides, which are

inherently biocompatible polymers usually extracted from natural

re-newable sources, can be functionalized for nanomedicine applications

Among many choices of semi-processed natural polymers,

carbox-ymethylcellulose (CMC) finds extensive use in biology, nutrition,

medicine, and pharmaceuticals CMC is a commercially available

cel-lulose derivative, which comprises unique physicochemical and

bio-chemical properties, including a remarkable water solubility in a wide

range of pH (e.g., at physiological conditions) CMC biopolymers

pos-sess amphiphilic behavior and reactive chemical groups (e.g., hydroxyl

and carboxylic), which permit their functionalization with

biomole-cules 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,

bio-medical, and pharmaceutical products Hence, polymer-drug

nanosys-tems 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

tar-geting, 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

lim-ited 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

nanos-tructures has emerged, encompassing the properties of semiconductor

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 na-nostructures 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 na-nohybrids composed offluorescent Cu-In-S/ZnS QDs can be synthesized

by a green aqueous colloidal process using CMC biopolymer simulta-neously as capping ligand and targeting macromolecule coupled to folic acid, and electrostatically complexed with DOX anticancer for produ-cing 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

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 sub-stitution 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, emis-sion spectra atλexc= 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 ac-cording toAggeryd and Olin (1985)

2.2 Synthesis, functionalization, and characterization nanoconjugates CIS and ZCIS quantum dots were synthesized via an aqueous pro-cess Under magnetic stirring, 2.0 mL of the indium solution (1 × 10−2 M) and 0.12 mL of copper solution (1 × 10−2M) 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 × 10−2M) was injected into theflask, 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 × 10−2M) was added into 42 mL of the CIS@CMC sus-pension After 1 min, 1.0 mL of sulfide solution (1 × 10−2M) 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 com-position 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'-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) crosslinking reaction andL-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

A.A.P Mansur, et al. Carbohydrate Polymers 247 (2020) 116703

(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

inter-actions 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

Beer-Lambert 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

fea-tures: ultraviolet-visible and photoluminescence spectroscopy

(steady-state, 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-buf-fered 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 afinal concentration of 2.5 nM of

ZCIS nanoparticles (ZCIS@CMC, ZCIS@CMC-FA, and

ZCIS@CMC-FA-DOX) and 5.0μM of DOX (ZCIS@CMC-FA-DOX) As references, CMC

solution and free DOX at thefinal 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

inter-nalization 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

TRITCfilters after exposing cell lines (MCF7 and TNBC) to samples for

30 min

All specific details and protocols related to the materials and

ex-perimental procedures are detailed in the Supplementary Material

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

disper-sions Thus, in this study, the comprehensive characterization of CMC

was conducted using several spectroscopic analyses and biological

as-says

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.,

car-boxylic/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

sub-stituted 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

moieties (COO−), which was maintained with the increase of pH (10.5) In the acidic medium (pH = 3.5), negatively charged carbox-ylate groups are protonated, forming carboxylic acid (R-COO−+ H+/ R-COOH), decreasing the ZP of the macromolecular system

3.2 Characterization of CIS and ZCIS nanoparticles UV–vis spectroscopy of CIS@CMC (Fig 2A(a)) nanoparticles pre-sented a featureless and broad absorption curve with band edge ex-tending 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 nano-colloids, these results supported the hypothesis that the ternary CIS quantum dots were effectively nucleated and stabilized by CMC poly-saccharide 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 en-ergy 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 Keyfindings are sum-marized 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 offluorescence 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 organicfluorophores (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 con-tinuous 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 absorptionfindings 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, insetFig 2A(a)) evi-denced the single-crystalline property CIS@CMC This feature is im-portant because it revealed the capability of CMC as a hard-base

A.A.P Mansur, et al. Carbohydrate Polymers 247 (2020) 116703

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

A.A.P Mansur, et al. Carbohydrate Polymers 247 (2020) 116703

composition of these nanoconjugates produced The XPS spectra

con-firmed 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)

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

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

A.A.P Mansur, et al. Carbohydrate Polymers 247 (2020) 116703

agent and a capping ligand for the nucleation and growth offluorescent

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

interac-tions occurring between the functional groups of the CMC ligand and

the inorganic nanocrystal The FTIR spectra at the range of 4000−2500

cm−1showed 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)), sig-nificant 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 andβ1-4 glycoside bond

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 (left-column) and (B) ZCIS@CMC (right (left-column)

A.A.P Mansur, et al. Carbohydrate Polymers 247 (2020) 116703

(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−1of 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

inter-actions 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

in-dicating 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 (Δν1and

Δν2) Based on the literature, Δν values obtained from the spectra

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 poly-saccharide played a relevant role in the nucleation, growth, and sta-bilization of the colloids The XPS results of CMC ligand are presented

inFig 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 poly-saccharide shell layer These FTIR and XPS results confirmed the in-teraction of CMC polymer with the nanoparticle surface that favors the blocking of surface trap states, which resulted in non-radiative re-combination 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 so-lution 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 li-gands, 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 ac-counted 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 dis-persed 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 (ap-proximately 100 %) of the polymeric shell around the pristine CIS in-organic core.Fig 6depicts a schematic representation of the interac-tions at interface based on the FTIR, XPS, ZP, and DLS results 3.3 Cancer nanotheranostic applications of core-shell nanohybrids For targeted-bioimaging and anticancer drug delivery, the con-jugation of folic acid to CMC biopolymer was performed in two stages: (I) coupling L-Arginine to ZCIS@CMC nanostructures; and (II) con-jugation 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

Fig 4 FTIR spectra in the range of (A) 4000–2500 cm−1and (B) 1800–800

cm−1for (a) CMC, (b) CIS@CMC, and (c) ZCIS@CMC

A.A.P Mansur, et al. Carbohydrate Polymers 247 (2020) 116703

steps related to the formation of the FA-modified polymer were

eval-uated 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

pre-sented Bands of COO− groups of CMC associated with asymmetric

(1592 cm−1) and symmetric (1418 cm−1and 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 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

A.A.P Mansur, et al. Carbohydrate Polymers 247 (2020) 116703

observed (Carvalho et al., 2019) Also, guanidino peaks of L-Arginine at

1675 cm−1and 1633 cm−1, and the bands at 1475 cm−1and 1455

cm−1associated with theeCH2groups of the aliphatic side chain were

detected After the second stageFig 7A(c), a relative increase of the

intensity of the bands of amides (–CONH-) was identified at 1540 cm−1

and 1240 cm−1confirming the reaction of carboxylates from glutamic

acid subpart of FA molecule with side-chain guanidino groups from L-Arginine, as the amino groups partially reacted at thefirst stage of the conjugation process (Psarra et al., 2017)

Fig 7B presented thefluorescent imaging features of pure DOX drug (a), pure FA (b), ZCIS@CMC (c), and ZCIS@CMC-FA-DOX complex (d),

at the excitation wavelength ofλexc= 375 nm DOX emission showed

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)

A.A.P Mansur, et al. Carbohydrate Polymers 247 (2020) 116703

its yellow-red fluorescence signature with a maximum at 592 nm

as-sociated with of quinonoid structure (Angeloni, Smulevich, &

Marzocchi, 1982) For FA, the presence of subunits, pterin and

4-ami-nobenzoyl 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

pre-served the DOX and FAfluorescence 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

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 nanos-tructures

The acellular in vitro drug release experiment of the ZCIS@CMC-FA-DOX complexes in comparison to ZCIS@CMC-FA-DOX in free form was performed by the dialysis method (PBS, pH = 7.4 ± 0.2) for 24 h to simulate phy-siological 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 accu-mulated drug achieved after 24 h This indicated that the release was

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

A.A.P Mansur, et al. Carbohydrate Polymers 247 (2020) 116703