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.

Carbohydrate Polymers 247 (2020) 116703 Contents lists available at ScienceDirect 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 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 – 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) https://doi.org/10.1016/j.carbpol.2020.116703 Received April 2020; Received in revised form 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 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 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 ± °C for h This suspension was left to cool down to room temperature and dialyzed for 24 h against 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 min, 1.0 mL of sulfide solution (1 × 10−2 M) was injected and thermally treated for h at 90 ± °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 Carbohydrate Polymers 247 (2020) 116703 A.A.P Mansur, et al 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 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 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 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 Carbohydrate Polymers 247 (2020) 116703 A.A.P Mansur, et al Fig 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 Carbohydrate Polymers 247 (2020) 116703 A.A.P Mansur, et al Fig (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) Carbohydrate Polymers 247 (2020) 116703 A.A.P Mansur, et al Fig (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 Carbohydrate Polymers 247 (2020) 116703 A.A.P Mansur, et al 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 depicts a schematic representation of the interactions at interface based on the FTIR, XPS, ZP, and DLS results + Fig 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 Carbohydrate Polymers 247 (2020) 116703 A.A.P Mansur, et al Fig 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 Carbohydrate Polymers 247 (2020) 116703 A.A.P Mansur, et al Fig 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 Carbohydrate Polymers 247 (2020) 116703 A.A.P Mansur, et al Fig (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 10 Carbohydrate Polymers 247 (2020) 116703 A.A.P Mansur, et al 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 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, 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 h of incubation with nanocarriers, where an augment of 50 % in killing activity in comparison to the unbound drug 11 Carbohydrate Polymers 247 (2020) 116703 A.A.P Mansur, et al Fig 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 with: (A) TNBC (FRα+) and (B) MCF7 cells (FRα-) (scale bar = 10 μm) 12 Carbohydrate Polymers 247 (2020) 116703 A.A.P Mansur, et al Fig 10 Illustration of the targeted drug-delivery process of fluorescent ZCIS@CMC-FA-DOX nanoarchitecture (not to scale) 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:https://doi.org/10.1016/j.carbpol.2020.116703 The financial support of this study was provided by the Brazilian Funding Research Agencies: Fundaỗóo de Amparo Pesquisa Estado de Minas Gerais – FAPEMIG (Grants: UNIVERSAL-APQ-00291-18; PROBIC-2018; PPM-00760-16); Coordenaỗóo de Aperfeiỗoamento de Pessoal de Nớvel Superior - CAPES (Grants: PROINFRA2010–2014; PROEX-433/2010; PNPD-2014-2019); Financiadora de Estudos e Projetos - FINEP (Grants: CTINFRA/PROINFRA 2008/2010/2011/ 2018); 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