Surfactants have been used as a tool to improve the properties of polymeric nanoparticles (NPs) and to increase the rate of hydrophobic drug release by means of these nanoparticles. In this context, this study evaluated the effect of lecithin on the characteristics of chitosan (CHI) and chondroitin sulfate (CS) nanoparticles, when applied in curcumin (Curc) release.
Carbohydrate Polymers 227 (2020) 115351 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol The role of the lecithin addition in the properties and cytotoxic activity of chitosan and chondroitin sulfate nanoparticles containing curcumin T Katiúscia Vieira Jardima, Joseilma Luciana Neves Siqueirab, Sônia Nair Báob, ⁎ Marcelo Henrique Sousaa, Alexandre Luis Parizec, a Green Nanotechnology Group, Universidade de Brasília, Brasília, DF 72220-900, Brazil Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, CampusUniversitário Darcy Ribeiro – Asa Norte, Brasília, DF 70910-900, Brazil c Polimat, Grupo de Estudos em Materiais Poliméricos, Departamento de Química, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-900, Brazil b A R T I C LE I N FO A B S T R A C T Keywords: Chitosan Chondroitin sulfate Lecithin Curcumin Polymeric nanoparticles Drug delivery Surfactants have been used as a tool to improve the properties of polymeric nanoparticles (NPs) and to increase the rate of hydrophobic drug release by means of these nanoparticles In this context, this study evaluated the effect of lecithin on the characteristics of chitosan (CHI) and chondroitin sulfate (CS) nanoparticles, when applied in curcumin (Curc) release CHI/CS NPs and CHI/CS/Lecithin NPs were prepared by the ionic gelation method, both as standards and containing curcumin Simultaneous conductimetric and potentiometric titrations were employed to optimize the interaction between the polymers NPs with hydrodynamic diameter of ∼130 nm and zeta potential of +60 mV were obtained and characterized by HRTEM; their pore size and surface area were also analyzed by BET method, DLS, FTIR, XPS, and fluorescence spectroscopy techniques to assess morphological and surface properties, stability and interaction between polymers and to quantify the loading of drugs The final characteristics of NPs were directly influenced by lecithin addition, exhibiting enhanced encapsulation efficiency of curcumin (131.8 μg curcumin per mg CHI/CS/Lecithin/Curc NPs) The release of curcumin occurred gradually through a two-stage process: diffusion-controlled dissolution and release of curcumin controlled by dissolution of the polymer However, the release of curcumin in buffer solution at pH 7.4 was achieved faster in CHI/CS/ Lecithin/Curc NPs than in CHI/CS/Curc NPs in vitro cytotoxic activity evaluation of the curcumin was determined by the MTT assay, observing that free curcumin and curcumin nanoencapsulated in CHI/CS/Curc and CHI/CS/Lecithin/Curc NPs reduced the viability of MCF-7 cells in the 72 h period (by 28.4, 36.0 and 30.7%, P < 0.0001, respectively) These results indicate that CHI/CS/Lecithin NPs have more appropriate characteristics for encapsulation of curcumin Introduction Curcumin (1,7bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene3,5-dione) is a yellow solid, classified according to the Biopharmaceutics Classification System (BCS) as a class II drug that is poorly water-soluble but highly permeable It is a phenolic compound that has methoxy and phenol groups in its chemical composition, which are responsible for its biological and pharmacological properties (Dai et al., 2018) Its potential in the prevention and treatment of various diseases, including cancer, has been extensively investigated in recent years, since it has antiproliferative and pro-apoptotic effects against several types of tumors, contributing mainly to the inhibition of tumor growth (Calaf, Ponce-Cusi, & Carrión, 2018) Recently, Siddiqui et al ⁎ (2018) showed that curcumin decreases the Warburg effect on several cancer cells (H1299, MCF-7, HeLa and PC3) Similarly, DavatgaranTaghipour et al (2017) presented experimental evidence and clinical perspectives that polyphenols such as curcumin are potentially capable of acting as chemopreventive and chemotherapeutic agents in different types of cancer In addition, the authors report that nanoformulations of natural polyphenols as bioactive agents, including resveratrol, curcumin, quercetin, epigallocatechin-3-gallate, chrysin, baicalin, luteolin, honokiol, silibinin and coumarin derivatives, in a dose-dependent manner, result in prevention and treatment of cancer However, Nelson, Dahlin, Bisson, Graham, Pauli & Walters (2017) reported in their study that although the activity and therapeutic utility of curcumin have increased the interest of scientists, no evidence of the therapeutic benefits Corresponding author E-mail address: alexandre.parize@ufsc.br (A.L Parize) https://doi.org/10.1016/j.carbpol.2019.115351 Received 11 February 2019; Received in revised form July 2019; Accepted 18 September 2019 Available online 21 September 2019 0144-8617/ © 2019 Elsevier Ltd All rights reserved Carbohydrate Polymers 227 (2020) 115351 K.V Jardim, et al and enhanced efficiency in the encapsulation of drugs (Shin, Chung, Kim, Joung, & Park, 2013; Yang, Dai, Sun, & Gao, 2018; Tsai, Chiu, Lin, Chen, Huang, & Wang, 2011) In this context, the objective of this study was to evaluate the effect of adding lecithin on the characteristics of chitosan (CHI) and chondroitin sulfate (CS) nanoparticles, prepared by the ionic gelation method, used for the controlled in vitro release of curcumin and to improve its cytotoxic activity in human breast tumor cells (MCF-7) of curcumin has been found The authors claim that curcumin has disadvantages as a candidate in the clinical setting, since it has low solubility in aqueous solutions, high decomposition rate in neutral or basic pH and susceptibility to photochemical degradation, which is also reported in other studies (Chuah, Roberts, Billa, Abdullah, & Rosli, 2014; Lim et al., 2018) However, considering these controversies regarding the therapeutic efficacy of curcumin, Heger (2017) suggests that the thousands of research papers and more than 120 clinical trials performed with curcumin should not be discarded; it is particularly worth further investigating its potential as a therapeutic agent In this context, several strategies have been evaluated to increase the biological activity of curcumin, mainly aiming for greater absorption and availability to tissues (Akbar et al., 2018) Several methods are described in the literature for improving the solubility of curcumin, such as: impregnation (Parize et al., 2009), liposomes (Li et al., 2018), copolymers and nanoemulsions (Akbar et al., 2018; Dai et al., 2018), chemical modifications in curcumin structure (Mohamed, El-Shishtawy, Al-Bar, & Al-Najada, 2017), and association in polymer nanoparticles (Jardim, Joanitti, Azevedo, & Parize, 2015), among others Polymeric nanoparticles appear as easy-to-prepare systems and increase the effectiveness of the treatment, due to the increased solubility and increased effectiveness of the drug Recently, nanoparticles formed from chitosan and chondroitin sulfate through ionic polyelectrolytic complexation have been reported as a promising alternative for the encapsulation and release of hydrophobic drugs, such as curcumin, since it is a simple and reversible process (Umerska, Corrigan, & Tajber, 2017) In addition, NPs formed through ionic crosslinking have the ability to protect the active substance against degradation and increase its bioavailability in a physiological environment (Tsai, Chen, Bai, & Chen, 2011) Chitosan can also be associated with biocompatible surfactants, such as lecithin, which promotes improvements in the properties of the polymer network that is formed, as well as an increase in the incorporation rate of the drug (Dammak & Sobral, 2018; Şenyiğit et al., 2017; Terrón-Mejía et al., 2018) Chitosan is a natural biopolymer obtained from the reaction of Ndeacetylation of chitin in alkaline medium It is represented as a copolymer of 2-amine-2-deoxy-D-glucose and 2-acetamide-2-deoxy-Dglucose, linked by β-type glycosidic bonds (1,4) It has a wide range of applications because it is biodegradable, biocompatible, bioadhesive and non-toxic (Biswas, Chattopadhyay, Sen, & Saha, 2015; TerrónMejía et al., 2018) When dissolved in aqueous acid solutions, pH < 6.2 has a positive charge in the −NH3+ groups, which facilitates their solvation in water and aggregation to polyanionic compounds, such as chondroitin sulfate, forming polyelectrolyte complexes (PECs) (Şenyiğit et al., 2017; Terrón-Mejía et al., 2018) Chondroitin sulfate belongs to the family of glycosaminaglans (GAGs), and it is characterized as an alternating copolymer of the monomers β(1,4)-D-glucuronic acid and β(1,3)-N-acetyl-D- galactosamine, which may be sulfated at the C4 or C6 carbons It has low toxicity, biocompatibility and specific biodegradability (Krichen et al., 2018) In addition, it can form polyelectrolyte complexes (PECs) through electrostatic interaction with positively charged substances, thus providing an optimal strategy to maintain CS in the solid state for use as a drug delivery system (Jardim et al., 2015; Gul et al., 2018; Tan, Selig, & Abbaspourrad, 2018) Nanoparticles based on PECs formed by biopolymers not often promote adequate dispersion, solubilization and bioavailability of drugs such as curcumin Thus, to promote greater stability and efficiency in the encapsulation of poorly soluble drugs, a coating with biocompatible compounds, such as lecithin, is required (Sun et al., 2015) Lecithin is a highly bioactive compound that consists of a glycerol backbone esterified with two fatty acids and a phosphate group, endowing it with strong potential for use in the food and pharmaceutical industries as an emulsifier, nutrition enhancer and carrier (Pawar & Babu, 2014) The nanoparticles prepared with lecithin and chitosan showed higher bioavailability, mucoadhesive property, storage stability Materials and methods Chitosan (99% purity) (medium molecular weight) with a molecular mass around 106 kg mol−1 and deacetylation degree ∼76.9% was determined by conductimetric titration (Alvarenga, 2011) Chondroitin 4-sulfate sodium salt (99% purity), originating from bovine trachea and curcumin (95% purity), originating from Curcuma Longa L., with purity of 98%, were obtained from Sigma-Aldrich (St Louis, MO, USA) Lecithin obtained from egg yolk (60% of L-α-Phosphatidylcholine – Sigma Aldrich Dulbecco's Modified Eagle Medium (DMEM) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) were obtained from Life Technologies (USA), and the line of human breast tumor cells (MCF-7) was obtained from the cell bank of Rio de Janeiro (BCRJ), Brazil The other reagents were of analytical grade and were used without prior purification 2.1 Sample elaboration The logarithmic values of the dissociation constant (pKa = −logKa) were determined for pure CHI and CS samples, by means of simultaneous potentiometric and conductimetric titrations as described by Farris, Mora, Capretti, and Piergiovanni (2012), to optimize conditions of interaction between the polymers during synthesis of NPs In this way, 50 mL of the CHI or CS solutions (0.1 wt%) were titrated with a 0.1 mol.L−1 HCl or NaOH solution, using a Metrohm 856 Conductivity Module with a 5-ring conductivity measuring cell (c =0.7 cm−1 with Pt1000) and a Metrohm 827 pH lab pHmeter Before titration, the pH of the CS and CHI solution were respectively adjusted to ∼2.0 and ∼7.0, using HCl and NaOH solutions Based on the pKa values, the speciation diagram of the mole fractions of the surface sites as a function of pH was constructed, thus establishing the ideal pH (5.5) for obtaining the nanoparticles The synthesis of CHI/CS and CHI/CS/Lecithin NPs was performed by the ionic gelation method (Fan, Yan, Xu, & Ni, 2012) For this, homogeneous solutions of CHI (1.0 mg/mL) and CS (1.0 mg/mL) were prepared in 0.1 mol.L−1 acetic acid solution and pH adjusted to ∼5.5 To obtain the CHI/CS NPs, 150 mL of CS solution was added slowly to 100 mL of CHI solution The CHI/CS/Lecithin NPs were obtained with the addition of 5.0 mL of 3.5% (w/v) of lecithin ethanol solution to 100 mL of CHI solution, and then 150 mL of the chondroitin sulfate solution was added slowly The lecithin-chitosan ratio was set at 1:20 (w/w) in the nanoparticles The formation of the NPs was conducted under constant magnetic stirring for 40 at 25 °C The encapsulation of curcumin into the CHI/CS NPs was carried out by adding an ethanolic solution of curcumin to the CHI solution prior to the interaction with the CS solution The following synthetic methodology is the same as that described in the previous paragraph in relation to the production of the CHI/CS NPs based on a previous study by Jardim et al (2015) A small amount of curcumin (∼30 mg) was added, and stabilized in 100 mL of the CHI solution, which was maintained at pH 5.5 After the curcumin had come into contact with the chitosan solutions, the 150 mL of CS solution at pH 5.5 was added slowly to the solution containing CHI/Curc The solutions were maintained under constant magnetic stirring for 40 at 25 °C, leading to the formation of CHI/CS/Curc NPs The incorporation of curcumin in the CHI/CS/ Lecithin NPs occurred prior to the addition of 5.0 mL of 3.5% (w/v) of lecithin ethanol solution to 100 mL of CHI solution at pH 5.5 and then Carbohydrate Polymers 227 (2020) 115351 K.V Jardim, et al were excited at 429 nm, and the emission spectra were recorded from 440 to 700 nm The relative fluorescence intensities were measured at λ = 540 nm and compared to a standard calibration curve To construct the calibration curve, a stock solution of 108 μmol.L−1 curcumin was prepared in a 80:20 (v/v) mixture of 0.1 mol.L−1 solution of phosphate buffer solution (pH 7.4):ethanol From the stock solutions, the working solutions were prepared at concentrations of 0.5 to 15 μmol.L−1 by dilutions of the stock solution in the phosphate buffer solution (pH 7.4):ethanol solution Each sample was assayed in triplicate, and the results were expressed as the amount of curcumin (in μg) per mg of nanoparticles Similarly, the encapsulation efficiency (EE%) was calculated as the ratio between the amount of drug entrapped in the nanoparticles and the initial amount of curcumin used to prepare the nanoparticle batch was added to curcumin (˜ 30 mg) and 150 mL of CS solution at pH 5.5 The solutions were also maintained under constant magnetic stirring for about 40 at 25 °C, leading to the formation of CHI/CS/Lecithin/ Curc NPs 2.2 Sample characterization The morphology and size of the prepared NPs was evaluated by high-resolution transmission electron microscopy (HRTEM) using a JEOL JEM-2100 microscope equipped with EDS, Thermo scientific For HRTEM analysis, the colloidal suspensions obtained were diluted in water in a ratio of 50 μL of the colloidal suspension of NPs to 100 μL of Type water A small aliquot of the resulting sample dilution (3 μL) was placed on a copper screen (400 mesh), covered with a carbon film and, before the measurements were taken, a solution of phosphotungstic acid (2.0% w/v) was applied to provide contrast for better visualization of NPs under the microscope Dynamic light scattering (DLS) (Nano-Zetasizer-ZS, Malvern Instruments) was used to determine the hydrodynamic diameter, the polydispersity index (PDI) and the zeta potential of the NPs as a function of pH For DLS analysis, the NPs were dispersed in water (about 0.01 wt%), sonicated for 10 and pH adjusted with 0.1 mol.L−1 of the HCl or NaOH solutions The surface area and pore volume distribution of the NPs were measured using a mass of 0.2 g sample at 77 K in AUTOSORB-1 equipment with a free space of about 16 cm³, cold free space around 48 cm³, equilibrium interval of 10 s, no low pressure dose and automatic degassing The stability of the nanoparticles was evaluated by monitoring the hydrodynamic diameter and the zeta potential, using Dynamic light scattering (DLS) equipment (Nano-Zetasizer-ZS, Malvern Instruments) The samples were kept at 37 °C in the form of aqueous suspension, and the measurements were performed in triplicate, using a diluted solution (0.01 wt%) of the samples, during the period of 90 days The interpretation of data was performed by cumulative analysis of the experimental correlation function, and hydrodynamic radius was calculated from the computed diffusion coefficients using the Stokes–Einstein equation (Eq 2) kT Rh = 6πηDt 2.4 Curcumin release profile The kinetics of curcumin release was performed by adapting the methodology described by Parize et al (2009) For this purpose, approximately 30 mg of curcumin-containing CHI/CS/Curc and CHI/CS/ Lecithin/Curc NPs were suspended in 30 mL of 0.1 mol.L−1 phosphate buffer solution at pH 7.4 The samples were kept under constant stirring (700 rpm) in a thermostat-controlled bath at 37.0 ± 0.1 °C The analysis was conducted for 240 h by means of measurements at predetermined time intervals, where a mL aliquot of the supernatant was analyzed on a Fluorolog-TSPC (Horiba-Jovine Ivone) fluorimeter with both slits of excitation, and emission monochromators were adjusted to 5.0 nm The samples were excited at 429 nm, and the emission spectra were recorded from 440 to 700 nm The amount of curcumin released was determined using the Curc calibration curves, which correlate the fluorescence intensity with the known concentration of curcumin (μmol/L) in the same solution where release kinetics was conducted The results obtained were presented as percentage of release of curcumin over time The release mechanism was analyzed by adjusting the release kinetics profiles, applying the Gallagher-Corrigan mathematical model (Gallagher & Corrigan, 2000) 2.5 Biological tests 2.5.1 Evaluation of in vitro cytotoxic activity For the cell culture, the human breast epithelial adenocarcinoma cell line (MCF-7) was routinely maintained in cell culture flasks (75 cm2) in an incubator (37 °C, 5% CO2 and 98% humidity) with 15 mL of DMEM cell culture medium supplemented with 10% (v/v) fetal bovine serum (FBS, Life Technologies, USA) and 1% (v/v) antibiotic solution (100 U/mL penicillin – 100 μg/mL streptomycin, Life Technologies, USA) and passaged every or days For the passage or preparation of the experiments, the cells were desalted with mL of 0.25% Trypsin-EDTA (Life Technologies, USA) and then inactivated with mL DMEM supplemented with 10% FBS and 1% antibiotic solution Cell viability was determined by the 3,4,5-dimethylthiazol-2,5-biphenyl tetrazolium bromide (MTT) assay, where MCF-7 cells (5 × 103 cells/well in 200 μL of DMEM) were seeded on 96-well plates and allowed to attach overnight Cells were then incubated with 200 μL/well of DMEM containing different concentrations (10 to 40 μmol.L−1) of CHI/CS and CHI/CS/Lecithin NPs (with or without curcumin) and free curcumin at 37 °C (5% CO2) The pH of the samples was adjusted to 7.4 with NaOH (1.0 mol.L−1) before being seeded with the cells After 24, 48 and 72 h of incubation, the treatment was withdrawn and then 150 μL of MTT solution (0.5 mg/mL in DMEM) was added in each well, and incubation was carried out for h at 37 °C (5% CO2) The culture medium was then aspirated, and 200 μL of dimethyl sulfoxide was added to dissolve the purple formazan crystals from viable cells The absorbance was determined using a spectrophotometer with a microplate reader at a wavelength of 595 nm (SpectraMax®, model M2, (1) where Dt is the diffusion coefficient, k is the Boltzmann constant, T is the absolute temperature, η is the dynamic viscosity and Rh is the particle diameter The FTIR spectra were recorded with KBr pellets in the region of 4000–400 cm−1 on a Varian FTIR spectrophotometer with a resolution of cm−1 By means of X-ray photoelectron spectrometry (XPS), strains were acquired in a SPECS SAGE HR 100 system spectrometer, with energy of 30 eV and 15 eV for analysis of the regions and of 285 eV for calibration of the binding energies of the peak C 1s The atomic percentage of the elements present on the surface of the NPs under study and their possible interactions were determined 2.3 Determination of curcumin loading In the quantification of curcumin, performed by the filtration/centrifugation technique (Sun, Me, Tian, & Liu, 2007), the free active was determined in the supernatant and the total active was measured after the complete dissolution of the samples To obtain the supernatant, 10 mg of nanoparticle-based samples were dispersed in 10 mL of an 80:20 (v/v) mixture of 0.1 mol.L−1 phosphate buffer solution (pH 7.4):ethanol After filtering through 0.2 μm PTFE filter, an aliquot of the supernatant was transferred to a quartz cell and analyzed in a Fluorolog-TSPC (Horiba-Jovine Ivone) fluorimeter Both slits of excitation and emission monochromators were adjusted to 5.0 nm The samples Carbohydrate Polymers 227 (2020) 115351 K.V Jardim, et al deprotonation of the −SO3H and −COOH groups, where the third zone is initiated In the third zone, there was a sharp increase in the conductivity due to the excess of OH− ions in the dispersion For CS, pKa1 = 2.5 (−SO3H) and pKa2 = 4.0 (−COOH) were estimated from titration data as described by Campos, Tourinho, Da Silva, Lara, and Depeyrot (2001) Thus, taking into account the pKa values obtained in the titrations, the molar fraction of polymer surface species was plotted against the pH, as shown in Fig 1C In these speciation curves, it was verified that CS presents three distinct superficial sites In an extremely acidic medium, protonated sulfonic and carboxylic groups (CS-SO3H−COOH) are observed With the progressive increase of pH, sequential deprotonation of the sulfonic and carboxylic groups occurs Thus, the surface of CS becomes negatively charged (−SO3− and −COO−), allowing complexation with positively charged species in neutral and alkaline pHs (Rodrigues, Cardoso, Da Costa, & Grenha, 2015) However, at these pHs the CHI remains deprotonated, as shown in speciation curves On the other hand, in an acidic medium the CHI is positively charged, because of the protonation of −NH2 groups (CHI−NH3+) Considering these speciation diagrams, at a pH halfway between the pKa of CHI and pK2 of CS (pH ∼5.5) most superficial sites of CHI will be positively charged and CS will be negatively charged Thus, the optimized interaction between CHI and CS can occur (Menegucci, Santos, Dias, Chaker, & Sousa, 2015) Also in Fig 1C, we observe the dependence of the zeta potential as a function of pH variation (2.0–12.0) of the CHI/CS NP dispersions From this curve, it was found that at pH < 7.0 zeta potential becomes positive and increases as pH decreases This is associated with the protonation of amine groups of CHI Above pH ∼7.0, an increasingly negative zeta potential was observed and associated with the deprotonation of −SO3H and −COOH groups This change in the zeta potential of the NPs confirms that the polymers are pH-responsive, i.e., the degree of ionization is significantly altered by virtue of a variation in the pH near the pKa value of their functional groups (Ganta, Devalapally, Shahiwala, & Amiji, 2008) This speciation study improved the ionic gelation method, favoring the formation of NPs with a narrow distribution and hydrodynamic diameter in the range of nanometers, as shown in Table Besides, the addition of lecithin resulted in the reduction of the hydrodynamic diameter and the PDI of the NPs (Table 1) The reduction in the hydrodynamic diameter of the nanoparticles after addition of lecithin may be related to the contribution of the attractive hydrophobic and electrostatic interactions that occur between the polymer and the surfactant This effect may be associated with the less effective overlap of electrostatic potentials around the polymer chain (Khan & Brettmann, 2019) In addition, the presence of the surfactant may cause changes in the behavior of the polymer in solution, such as surfactant-induced thickening, surfactant-induced swelling or compaction, surfactant-induced phase separation, among other effects (Silva, Antunes, Sousa, Valente, & Pais, 2011) Banik, Hussain, Ramteke, Sharma, and Maji (2012) also suggest that the surfactant may decrease the solubility of chitosan, favoring the formation of small particles On the other hand, an increase in the zeta potential of lecithin-containing NPs was observed due to the amine headgroup of choline present in the surfactant structure, which increases the positive charge density on the surface of NPs (Cheng, Oh, Wang, Raghavan, & Tung, 2014) The positive zeta potential with a relatively high modulus value (at pH ∼5.5) efficiently promoted curcumin encapsulation (Table 1) However, it was also observed that the amount of encapsulated curcumin was higher in CHI/CS/Lecithin/Curc NPs (131.8 μg/mg) than in CHI/CS/Curc NPs (118.4 μg/mg) This occurs because the negatively charged polar portion (phosphate groups) of the lecithin interacts with the −NH3+ groups of CHI, leading to the preliminary formation of a vesicle The self-assembling of phospholipid with the polymer leaves the fatty-acid positively-charged polar chains available to interact with the hydrophobic part of curcumin (Taner et al., 2014) In addition, the Molecular Devices, USA) This absorbance reading is directly proportional to the number of live cells in the culture The percentage of cell viability was presented as the percentage of the absorbance of treated cells to the absorbance of non-treated cells (100% × (absorbance of treated cells / absorbance of non-treated) 2.5.2 Internalization study of samples in MCF-7 cells For the analysis of sample internalization, × 106 MCF-7 cells/well were seeded on 6-well culture plates and, after adhesion, the cells were exposed to 40 μmol.L−1 free curcumin, CHI/CS/Curc NPs and CHI/CS/ Lecithin/Curc NPs for 24 h The desalted cells in microtubes were then washed with PBS and fixed with Karnovsky's solution (2.0% glutaraldehyde, 2.0% paraformaldehyde, 5.0 mmol CaCl2, 3.0% sucrose buffered in 0.1 mol.L−1 sodium cacodylate, at pH 7.2) for h at °C After fixation, the cells were washed in the same buffer and post-fixed for 30 in 1% osmium tetroxide, 0.8% potassium ferricyanide and 5.0 mmol CaCl2 in 0.1 mol.L−1 sodium cacodylate buffer Cells were washed twice with ultrapure water and then stained with 0.5% uranyl acetate overnight at °C The samples were washed twice with ultrapure water and dehydrated in increasing acetone gradient (50–100%) for 10 each and included in Spurr resin The ultrafine sections were obtained with an ultra-microtome (Leica, UCT, AG, Vienna, Austria) and analyzed in a JEOL JEM-2100 transmission electron microscope equipped with EDS, Thermo scientific 2.5.3 Statistical analysis All results are from three independent experiments and expressed as the mean ± SD The difference between the effect of the treated compound compared with the control values was verified by analysis of variance (ANOVA) and Tukey's post hoc test using the program GraphPad Prism® 5.0 The values that were significantly different from the control at P < 0.05 are indicated in the Figures by an asterisk Results and discussion Chitosan and chondroitin sulfate are polyfunctional polymers (with −NH2 and −SO3H and −COOH groups, respectively) that can be ionized in aqueous medium, where chitosan is represented by CHI-NH3+/ CHI-NH2 and chondroitin sulfate as CS−COOH−SO3H/ CS−COO−−SO3− in their protonated/deprotonated forms Considering that ionic crosslinking is based on the attractive electrostatic interaction between the CHI and CS polymers, it will be governed by the number of ionizable surface sites of the polymers, which depends directly on the pH of the dispersions (Maldonado, Terán, & Guzmán, 2012) Thus, before the preparation of the nanoparticles, the speciation profiles were constructed in order to determine the ideal pH to optimize the attractive interaction between the surface of the CHI and the CS polymers, by means of their pKa values obtained by simultaneous potentiometric and conductometric titrations (Fig 1) In the CHI titration (Fig 1A) two distinct zones were observed In zone 1, before adding the titrant (HCl), the pH of the CHI dispersion is ∼7.2 and the −NH2 groups are expected to be substantially deprotonated/discharged As the titrant is added, the protonation of NH2 groups occurs and CHI becomes positively charged Conductivity of CHI solution, low before adding the titrant, sharply increases at the equivalence point (pH 4.7) as an excess of titrant is added (zone 2) (Farris et al., 2012) Cross-linking conductometric and potentiometric data, a pKa = 6.3 was estimated for CHI In the titration curves of the CS (Fig 1B), three zones were observed In the first zone, as the titrant (NaOH) is added, a reduction in the conductivity is due to the neutralization of the excess of H3O+ ions (from HCl, used to adjust the initial pH), reaching the first point of equivalence (pH ∼1.9) In the second zone, the variation of conductivity is due to the deprotonation of the sulfonic and carboxylic groups (i.e [H3O+] increasing) and [Na+] variation, from the titrant (Scordilis-Kelley & Osteryoung, 1996) Thus, the second equivalence point (pH ∼7.4) was reached after the complete Carbohydrate Polymers 227 (2020) 115351 K.V Jardim, et al Fig Potentiometric (●) and conductometric (○) titration: CHI (A) and CS (B) Speciation diagram of the surface of CHI ( CHI-NH3+ and CHI-NH2) and CS ( CS-SO3H−COOH, CS-SO3−−COOH and CS-SO3−−COO−) (C) Representation of the chemical interactions between CHI and CS in obtaining the NPs by ionic gelation interfacial tension, resulting in the increase of the surface area and consequently the reduction of the size of the nanoparticles (Wen, Wen, Wei, Zushun, & Hong, 2012) This indicates that the selective cavities of NPs were more exposed after the addition of lecithin, allowing a greater encapsulation of curcumin In addition, it was verified that the increase in surface area is directly proportional to the increase in the mean volume of pores; however, there is an inverse correlation between the surface area, the hydrodynamic and pore diameters in all NPs (Marturano et al., 2017) In the NPs containing curcumin, it was observed that the values of surface area and mean volume of pores were relatively lower, when compared to the values obtained for NPs without curcumin, indicating the encapsulation of the drug, which causes changes in the characteristics of NPs (Silva, Fideles, & Fook, 2015) In prolonged periods of storage, NPs of this kind of material tend to form agglomerates (Kumari, Yadav, & Yadav, 2010) It is therefore a great challenge to produce systems that keep their colloidal stability for use of the lecithin in the internal phase increased the wettability of the solid particles of the drug, facilitating its adsorption on the nanoparticles (Chen et al., 2016) After encapsulation of the curcumin, an increase in the hydrodynamic diameter, the PDI and the zeta potential of the NPs was observed, as shown in Table This indicates the success in the encapsulation of the drug, which causes changes in the characteristics of the NPs (Marturano, Cerruti, Giamberini, Tylkowski, & Ambrogi, 2017) From the adsorption/desorption isotherms of N2 obtained by BET (data not shown) the presence of micropores (pore diameter < 20 Å) in the NPs was confirmed (Sing et al., 1985) The formation of the micropores may be related to the cross-linked bonds established between the biopolymers during the synthesis process (Lu, Le, Zhang, Huang, & Chen, 2017) From the BET results (Table 1) it was also observed that an increase occurred in the surface area of the NPs after addition of lecithin surfactant The presence of the surfactant reduces the Table Values for the size, PDI and zeta potential of the CHI/CS and CHI/CS/Lecithin and encapsulation efficiency (EE%) of curcumin Samples CHI/CS CHI/CS/Lecithin CHI/CS/Curc CHI/CS/Lecithin/ Curc a Size (nm)a 111.7 102.2 154.6 126.2 ± ± ± ± 1,8 1,5 1.3 1.6 PDIa 0.299 0.224 0.391 0.339 Zeta potential (mV)a ± ± ± ± 0.03 0.02 0.02 0.02 58.2 60.7 59.7 60.4 ± ± ± ± 1.3 1.1 1.1 1.2 BET analyses Curcumin Loading Surface area (m2/g) Pore volume (cm3/g) Pore diameter (Å) Amount of curcumin encapsulated (μg/mg)a Encapsulation efficiency (%EE)a 28.9 33.8 19.5 23.6 0.043 0.054 0.029 0.035 13.1 11.5 17.6 15.4 – – 118.4 ± 0.1 131.8 ± 0.2 – – 78.6 ± 0.4% 87.5 ± 0.5% Mean ± SD, n = Carbohydrate Polymers 227 (2020) 115351 K.V Jardim, et al Fig Stability of the NPs at 37 °C for the period of 90 days in function of the hydrodynamic diameter (A) and Zeta potential (B) for: (●) CHI/CS NPs; ( ) CHI/CS/Lecithin NPs; ( ) CHI/CS/Curc NPs and ( ) CHI/CS/Lecithin/ Curc NPs a long time Taking this into account, the stability of NP suspensions stored at 37 °C was monitored relative to the hydrodynamic diameter and zeta potential during the 90-day period The pH of samples was also monitored in this period and varied from 5.5 to 6.0 The results obtained are shown in Fig 2, where it can be verified that the zeta potential of NPs remained stable and positive, with a high modulus value (> +40 mV), as expected at this pH, indicating that there are large repulsive forces in the system, reducing the possibility of NP aggregation (Tsai, Chen et al., 2011) The CHI/CS/Lecithin NPs did not show statistically significant variations in relation to the hydrodynamic diameter during the 90-day period However, the CHI/CS NPs presented an increase of approximately 30% in relation to the original hydrodynamic diameter of the NPs over this period These results show that the addition of the surfactant reduces the flocculation of the particles, giving greater stability to the NPs Similar results were obtained for NPs containing curcumin As observed in the transmission electron micrograph displayed in Fig 3, the morphology of the NPs developed in this study corresponds to a compact structure with a tendency to exhibit a spherical shape, as has been described for many formulations of polysaccharide-based nanoparticles prepared by polyelectrolyte complexation (Hu, Chiang, Hong, & Yeh, 2012) However, the particle size observed with TEM (∼35 nm) was smaller compared to the result determined by DLS This discrepancy can be most likely explained as the shrinking of the nanoparticles during the drying process prior to the TEM observation The inset of Fig (corresponding to the micrograph of CHI/CS/Lecithin/ Curc NPs) shows a compact lipid nucleus surrounded by a contrasting layer of chitosan and chondroitin sulfate, confirming the presence of the polymers as the outmost layer surrounding the curcumin–lecithin complex, as the result of expected electrostatic interaction, based on opposite charges Similar results were observed by (Sonvico et al., Fig FTIR spectra for a) CHI; b) CS c) CHI/CS NPs; d) CHI/CS/Lecithin NPs, e) CHI/CS/Curc NPs, f) CHI/CS/Lecithin/Curc NPs and g) Curcumin 2006; Souza et al., 2014) The FTIR spectra (Fig 4) present the main bands of the chemical groups present in the NPs and their possible interactions In the spectrum of all the NPs a band at 1020 cm−1 assigned to the groups NH3+−SO3− was observed, indicating the interaction between the polymers (Jardim et al., 2015) In addition, the characteristic bands of chitosan, such as 1377-1257 cm-1 relative to the C–N bond, 1153 cm−1 attributed to the CeOeC bond of β 1–4 glucose, and 1072-1029 cm−1 due to the angular deformation of the amine group, were observed in the spectra of the NPs The presence of chondroitin sulfate was also confirmed in the spectra of the NPs by the observation of the bands: 1238-1060 cm−1and 856 cm−1 assigned to the S]O and CeOeS bonds, respectively In the CHI/CS and CHI/CS/Curc NPs spectra (Fig 4c and 4e) a shift was observed in the bands at 1658 cm−1 to 1639 cm−1 assigned to the amide I and at 1593 cm−1 to1559 cm−1 related to the angular deformation of the amine group This last shift indicates that the NH2 group in the NPs is in the form of NH3+ (Guilherme et al., 2010; Parize, Stulzer, Laranjeira, Brighente, & Souza, 2012) In the spectra of the NPs containing curcumin (Fig 4e and 4f) the band shift at 1593 cm−1 to 1558 cm−1 relative to NH2 of CHI was observed, indicating the interaction between the amine group of CHI and the phenolic group of curcumin The stretches at 3451 cm−1 assigned to the phenolic −OH group at 1620 cm−1 relative to the C]O bond of the conjugated ketone were also observed at 1562-1420 cm−1 related to the C]C bond of the aromatic ring, at 1380 cm−1 referring to Fig TEM images of CHI/CS/Lecithin/Curc NPs with different magnifications Histogram of particle diameters is shown in lower-right inset Carbohydrate Polymers 227 (2020) 115351 K.V Jardim, et al curcumin from the CHI/CS/Curc and CHI/CS/Lecithin/Curc NPs can be seen, performed at 37 °C in phosphate buffer solution at pH 7.4 during the 240 h period In this study, pH 7.4 was used as a stimulus for the release of curcumin, simulating physiological pH At pH 7.4, the −NH2 groups of chitosan (pKa = 6.3) are not ionized, while the −COOH and −SO3H groups of the chondroitin sulfate (pKa = 2.6 and 4.5) and phenolic groups of curcumin (pKa = 8.3) are deprotonated Thus, there is an increase in the density of negative charges, resulting in an aniontype electrostatic repulsion between the −COOH and −SO3H groups of the chondroitin sulfate and phenolic groups of curcumin This electrostatic repulsion associated with the reduction of the interaction force between CHI and CS destabilizes the NPs that are formed As a consequence, the curcumin molecules acquire greater mobility, thus facilitating their release into the environment (Yang et al., 2010) However, several mathematical models have been developed and studied to understand the release behavior of drugs from release systems (Pal, Singh, Anis, Thakur, & Bhattacharya, 2013) In this study, the mathematical model of Gallagher-Corrigan (Gallagher & Corrigan, 2000) (Eq 2) was applied in order to elucidate the mechanism by which curcumin is released from the NPs Table Chemical composition of the surface (in % at) obtained by XPS for nanoparticles and their constituents Samples C (% at) O (% at) N (% at) S (% at) P (% at) O/C (%) CHI CS Curcumin Lecithin CHI/CS CHI/CS/Lecithin CHI/CS/Curc CHI/CS/Lecithin/ Curc 54.5 50.2 58.1 52.0 52.2 47.3 41.2 35.6 39.7 41.8 41.9 42.3 40.7 43.1 52.7 55.4 5.8 4.9 – 2.3 4.5 5.4 3.7 4.9 – 3.1 – – 2.6 2.2 2.4 2.0 – – – 3.4 – 2.0 – 2.1 0.7 0.8 0.7 0.8 0.7 0.9 1.3 1.6 the CH3 groups and at 1070 cm−1 referring to the CeOeC vibration of the ether These bands were also observed in the curcumin spectrum (Fig 4g) (Anitha et al., 2011; Jardim et al., 2015; Şenyiğit et al., 2017) The presence of the band at 1593 cm−1 corresponding to the deformation of the amine group from CHI was not observed in Fig 4d and 4f, indicating the interaction of the NH2 groups of CHI with the phosphate groups of lecithin in an acid medium In the spectra of the lecithin-containing samples (Fig 4d and 4f), the following bands were observed: 3423 cm−1 and 3437 cm−1 were attributed to the stretches of the amine group; 1705 cm−1 corresponded to the carbonyl of the fatty acids present in lecithin, and the bands at 1053 cm−1 were related to the vibration of the phosphate group (Şenyiğit et al., 2017) The NPs were also analyzed by XPS to characterize their surface chemically and to evaluate the possible interactions between the polymers (Table 2) The composition obtained for CHI (54.5% C, 39.7% O and 5.8% N) is in agreement with the data obtained in the XPS analysis performed by Rodrigues, Da Costa, and Grenha (2012) The elemental composition of CS was found (50.2% C, 41.8% O, 4.9% N and 3.1% S) In the NPs only the elements carbon (C), oxygen (O), nitrogen (N) and sulfur (S) present in the chemical structures of their precursors were identified, indicating the absence of contamination during the synthesis process However, in NPs containing lecithin, the presence of phosphorus (P) and an increase in the atomic percentage of N was observed, indicating the contribution of the surfactant in the NPs The encapsulation of curcumin in the samples was further confirmed based on the increase in the O/C atomic mass ratio in the NPs containing curcumin CHI/CS/Curc NPs (1.3%) and CHI/CS/Lecithin/Curc NPs (1.6%) when compared to NPs without curcumin: CHC/CS NPs (0.7%) and CHI/CS/Lecithin NPs (0.9%) In Fig 5, the general aspect of the in vitro release curves obtained for e k2 t − k2 tm ⎤ ft = fB (1 − e−k1 t ) + (1 − fB ) ⎡ ⎢ + e k2 t − k2 tm ⎥ ⎦ ⎣ (2) Where ft is the fraction of the drug released at time t, fB is the maximum fraction of drug release, k1 is the release constant at the first stage, tm is the maximum release time and k2 is the release constant during the degradation of the polymer According to the mathematical model described by GallagherCorrigan the release of the drug from polymeric systems occurs in a two-stage process In the first stage (k1) a rapid release occurs due to the dissolution of the drug molecules present on the surface of the polymer matrix, and then slower release occurs due to the degradation of the polymer matrix (k2) (Gallagher & Corrigan, 2000) Thus, based on the Gallagher-Corrigan model, note that for the CHI/CS/Curc NPs, the values of k1 and k2 obtained were equal to 0.15 and 0.02, respectively For the CHI/CS/Lecithin/Curc NPs, the values of k1 and k2, obtained were: 0.26 and 0.007, respectively The first stage reflects the dissolution of curcumin in the medium, controlled by diffusion, observing thus a more accelerated release of curcumin In the second stage, the percentage of release of the curcumin is slower, probably because it depends on the dissolution of the polymer over time The results clearly show the difference in the release profile of curcumin caused by the addition of lecithin Although release of the curcumin occurs gradually in both NPs, the release percentage is higher for CHI/CS/Lecithin/Curc NPs in the first stage (k1), which can be attributed to the higher mean pore volume and the higher percentage of curcumin encapsulation in these nanoparticles (131.8 μg/mg = 87.5%), facilitating the diffusion of curcumin to the medium The viability of the MCF-7 human breast tumor cells from free and nanoencapsulated curcumin evaluated by the MTT assay (Fig 6) was significantly reduced (P < 0.0001), exhibiting dose-dependent cytotoxic activity, with increased concentration of curcumin from 10 to 40 μmol.L−1 at 24, 48 and 72 h of incubation When analyzing the effect of free curcumin, a decrease in cell viability was observed, by 50.0, 42.0 and 28.4% (P < 0.0001) when used in the concentrations of 10, 20 and 40 μmol.L−1, respectively, in the 72 h period In this same period, the addition of 10 μmol.L−1 of CHI/ CS/Curc and CHI/CS/Lecithin/Curc NPs reduced the viability of the MCF-7 cells by 56.4 and 58.0% (P < 0.0001), respectively When adding 20 μmol.L−1 of CHI/CS/Curc and CHI/CS/Lecithin/Curc NPs there was a reduction of 45.0 and 40.8% (P < 0.0001), respectively, in the viability of the cells With the addition of 40 μmol.L−1 of CHI/CS/ Curc and CHI/CS/Lecithin/Curc NPs, the cellular viability was reduced to 36.0 and 30.7% (P < 0.0001), respectively, when compared to the control group Fig Cumulative release of the curcumin from developed ( ) CHI/CS/Curc NPs and ( ) CHI/CS/Lecithin/Curc NPs at 37 °C in phosphate buffer solution at pH 7.4 for 240 h Carbohydrate Polymers 227 (2020) 115351 K.V Jardim, et al Fig Percentage of cell viability of MCF-7 after 24 h, 48 h and 72 h of incubation Viability assay by MTT Ultrapure water was used as negative control Significantly different from the control: *P < 0.0001 Fig Transmission electron micrographs obtained for MCF-7 cells treated with 40 μmol.L−1 of: (A) Control, (B) Curcumin and (C) CHI/CS/Lecithin/Curc NPs for 24 h with different magnifications Abbreviations: Nucleus (N); Mitochondria (M); Autophagic vesicle (arrow); Aggregation chromatin (arrow head closed); Apoptotic bodies (arrow head cast) released through the CHI/CS/Lecithin/Curc NPs were internalized in the cytoplasm of the MCF-7 cells, thus presenting signs of cytotoxicity The main ultrastructural changes in all treated groups were chromatin aggregation, mitochondrial denaturation, autophagy vesicle and apoptotic body formation, as well as cytoplasmic compartments, swelling and disappearance of mitochondrial cristae The therapeutic potential of curcumin as a cytotoxic agent has been extensively studied in recent years Several studies show that curcumin has distinct cytotoxicity profiles, depending on the cellular tissue and its concentration (Hanahan & Weinberg, 2011) In relation to human breast cancer, studies carried out by Bayomi et al., 2013; Bozta et al., 2013; Zhi-Dong et al., 2014 demonstrated that curcumin is an antiproliferative, cytotoxic and anti-metastatic agent The authors also evaluated the cytotoxic activity of free and encapsulated curcumin in nanoparticles in this cell line, obtaining profiles of cellular viability similar to those obtained in our study The most significant reduction in MCF-7 cell viability was observed over the 72 h period due to the prolonged exposure of free and nanoencapsulated curcumin in the cells, since the release of curcumin from the formulations occurred within the first 24 h of treatment In addition, increased inhibition in MCF-7 cell growth was observed for CHI/CS/Lecithin/Curc NPs Thus, this system represents a promising candidate for drug carrier for the treatment of breast cancer, since it has increased the therapeutic efficacy of curcumin In fact, the interaction with the biological medium of free and nanoentrapped curcumin is expected to be different, since the cellular uptake pathway of the nanoparticles is different from that of free drugs (Ahn, Seo, Kim, & Lee, 2013) While the free curcumin has direct contact with the cell, facilitating its diffusion into the membrane, the nanoparticles containing curcumin penetrate the cell by endocytosis, and the drug is liberated gradually to the medium, leading to a reduction in cytotoxic effect However, it is noteworthy that nanoparticles can be accumulated in tumor regions that exhibit abnormal vascularization and low lymphatic drainage (effect of improved permeability and retention - EPR) When accumulating in the area of interest, these nanoparticles should not only gradually release a high amount of the drug at the target site, but also minimize side effects in normal tissues and, as a result, decrease systemic toxicity (Hiroshi, Hideaki, & Jun, 2013) In the control experiments performed to evaluate the toxicity of the release system in the 24, 48 and 72 h periods (data not shown), it was observed that the addition of CHI/CS and CHI/CS/Lecithin NPs without the presence of curcumin did not alter the viability of the MCF-7 cells It was 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an increase in the atomic percentage of N was observed, indicating the contribution