dodecyl sulfate nanocarrier for a pyrazoline with antileukemic activity

Materials Science & Engineering C 105 (2019) 110051

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A new and efficient carboxymethyl-hexanoyl chitosan/dodecyl sulfate nanocarrier for a pyrazoline with antileukemic activity

T

Andrés Felipe Chamorro Rengifoa, , Natalia Stefanesb, Jessica Toigoc, Cassiana Mendesd, Maria C. Santos-Silvab, Ricardo J. Nunesc, Alexandre Luis Parized, Edson Minattia ⁎

a

Laboratório de Polímeros e Surfactantes, Departamento de Química, Centro de Ciências Físicas e Matemáticas, Universidade Federal de Santa Catarina, Campus Reitor João David Ferreira Lima, s/n - Trindade, Florianópolis, SC 88040-900, Brazil b Laboratório de Oncologia Experimental e Hemopatias, Centro de Ciências da Saúde, Universidade Federal de Santa Catarina, Campus Reitor João David Ferreira Lima, s/ n - Trindade, Florianópolis, SC 88040-900, Brazil c Laboratório de Estrutura e Atividade, Departamento de Química, Centro de Ciências Físicas e Matemáticas, Universidade Federal de Santa Catarina, Campus Reitor João David Ferreira Lima, s/n - Trindade, Florianópolis, SC 88040-900, Brazil d Grupo de Estudo em Materiais Poliméricos, Departamento de Química, Centro de Ciências Físicas e Matemáticas, Universidade Federal de Santa Catarina, Campus Reitor João David Ferreira Lima, s/n - Trindade, Florianópolis, SC 88040-900, Brazil

ARTICLE INFO

ABSTRACT

Keywords: Chitosan Nanocapsules Pyrazoline Antitumor activity Leukemia

We describe herein a chitosan nanocarrier for drug delivery applications obtained through the self-assembly of carboxymethyl-hexanoyl chitosan and dodecyl sulfate (CHC-SDS). Nanocapsules with spherical morphology were obtained in phosphate buffer at pH 7.4. These CHC-SDS nanocapsules showed no toxicity toward Jurkat cells (acute lymphoblastic leukemia) and were used to encapsulate a new pyrazoline (H3TM04) with antileukemia activity. The samples were characterized by dynamic light scattering (DLS) and Laser Doppler MicroElectrophoresis. The encapsulation efficiency was higher than 96% (293.6 μg mL−1) and the H3TM04-loaded nanocapsules (CHC-SDS-H) had a negative surface charge (−29.8 ± 0.7 mV) and hydrodynamic radius of around 84 nm. For the first time, CHC-SDS-H were formed and the antitumoral cancer activity was proved. The in vitro assays showed the controlled release of H3TM04 from the CHC-SDS-H nanocapsules in phosphate buffer pH 7.4. The H3TM04 release data were described by the power law model, indicating that H3TM04 delivery occurred via an erosion mechanism. The cytotoxicity assays with Jurkat and K-562 cells (acute myeloid leukemia) demonstrated that the CHC-SDS-H nanocapsule decreases the half maximal inhibitory concentration (IC50). The study showed that CHC-SDS nanocapsules represent a promising nanocarrier for pyrazoline derivates that could be applied in leukemia therapy.

1. Introduction Chemotherapy is the most common treatment for cancer. However, its effectiveness is limited by the relatively low amount of the therapeutic drug that reaches the tumor tissue and/or specific organs [1,2]. Reports in the literature show that some anticancer drugs, such as doxorubicin and cyclophosphamide, have serious side effects and high toxicity, the principal adverse effect being cardiotoxicity [3,4]. Nanoparticle drug delivery systems have been explored to overcome these drawbacks and optimize the controlled and sustained release [5,6]. However, the accumulation of nanoparticles in tumor tissues, low encapsulation efficiency and poor cell internalization has limited the development of effective cancer treatments [7]. Some important properties of nanoparticles, such as surface roughness, surface charge, ⁎

chemical composition and dispersibility have a direct influence on their internalization by cells through endocytose and consequently the drug absorption [8]. Hollow nanocapsules are a nano-vesicular system, where the drug is either confined to a inner reservoir or the lyophobic domains of the polymer shell or coating [9]. The use of polymers to obtain nanocapsules, with high biocompatibility and reproducibility, is reported in the literature, [9–11,14[15]. In addition, these nanocapsules can encapsulate large amounts of drug in their cavities, promote a controlled and sustained delivery and also protect the drug from the biochemical environment when in the body [9,12]. A previous example for the use of nanocapsules formed from polymers tested for antitumor activity is the work of Pereira and co-workers [12], who studied the antitumor activity of usnic acid encapsulated in

Corresponding author. E-mail address: [email protected] (A.F. Chamorro Rengifo).

https://doi.org/10.1016/j.msec.2019.110051 Received 26 May 2019; Received in revised form 18 July 2019; Accepted 2 August 2019 Available online 19 August 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.

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nanocapsules of poly (lactic co-glycolic acid), where the nanocarriers increased the tumor inhibition by 26.4% compared with the free usnic acid. In another study, nanocapsules of poly(H2NPEGCA-co-HDCA) copolymer were used to encapsulate 4-(N)-stearoyl gemcitabine and tested against human cancer cells. However, the active incorporation in the nanocapsules did not change the IC50 compared to the free active compound [13]. According to these reports, nanocapsules provide controlled release delivery and protect the drug from degradation. Despite the several advantages when using these nanocapsules, the delivery efficiency continues to be a challenge for their biomedical application [10,13]. Biopolymers are an interesting approach to overcome this limitation. Chitosan, an aminopolysaccharide, is one of the most abundant natural polymers[15]. It has been widely studied in biomedical applications due to its properties, including biodegradability, biocompatibility, low toxicity and bioadhesion [16–18]. However, the low aqueous and low organic solubility of chitosan has limited its use in drug delivery systems [17]. The chemical modification of chitosan can be tailored to alter the solubility of the natural polymer in water. Synthetic methods to obtain hydrophilic and hydrophobic chitosan derivatives have been reported [17,19]. The insertion of carboxyl and alkyl groups in the chitosan backbone can increase its solubility in water. Moreover, amphiphilic chitosan can self-assemble to form nanoparticles in aqueous solution, allowing its application as a nanocarrier [17]. There are several reports in the literature on amphiphilic chitosan [12,20,21]. However, there are less examples of hollow nanocapsules obtained from self-assembled amphiphilic chitosan applied in drug delivery. Kun-Ho Liu and co-workers used nanocapsules of carboxymethyl hexanoyl chitosan to encapsulate doxorubicin with an encapsulation efficiency of 46.8% [11]. Despite the acceptable results, the researchers did not investigate the nanocapsule cytotoxicity in order to evaluate the feasibility of the use of these kinds of hollow nanocapsules in biological applications. Moreover, the study concluded that a high degree of substitution of hexanoyl groups promoted agglomeration of the nanocapsules in aqueous solution. This hinders the biomedical application of these nanocarriers, since larger particles cannot be internalized by the targeted cells [21]. In the study reported herein, we produced nanocapsules of O,Ncarboxymethyl-hexanoyl-chitosan (CHC, Fig. 1A) and sodium dodecyl sulfate surfactant (SDS) by noncovalent association from the intra- or/ and intermolecular interaction between hydrophobic segments in aqueous solution. The samples were evaluated based on dynamic light scattering (DLS) analysis, zeta potential values and transmission electron microscopy (TEM) images. Pyrazoline H3TM04 ((1-(5-(naphthalen-2-yl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1yl) ethanone)) (Fig. 1B), a chalcone derivate, was used as a model drug with antileukemia action to investigate the potential for the use of CHCSDS nanocapsules as a drug carrier. The cytotoxicity H3TM04

encapsulated into CHC-SDS-H nanocapsules were then, for the first time, tested against leukemic cells. In vitro assays to determine the drug release behavior were also performed. 2. Materials and methods 2.1. Materials Chitosan of low molecular weight (50.000–190.000 g mol−1 according to the manufacturer) with a deacetylation degree of 75% (measured by 1H NMR [22]), sodium dodecyl sulfate (SDS) (≥99%), 3,4,5-trimethoxyphenylphenone (98%), hydrazine hydrate (70–80%), naphthaldehyde (98%), 2-propanol (≥99.4%), acetonitrile (≥99.8%), methanol (≥99.8%), dioxane (≥99.8%), sodium hydroxide (≥97%), chloroacetic acid (≥99%), hexanoyl anhydride (≥99%), sodium phosphate monobasic (≥99%), sodium phosphate dibasic, (≥99%), uranyl acetate (98%) and the dialysis membrane (approximate molecular weight cut off 3.500 g mol−1) were all purchased from SigmaAldrich (USA). All chemicals were used without further purification. Milli-Q water was used to prepare all solutions. 2.1.1. Procedure for H3TM04 synthesis According to our previous report [23], the pyrazoline was synthetized from 3,4,5-trimethoxyphenyl chalcone, which was obtained by aldol condensation of 3,4,5-trimethoxyphenyl phenone with naphthaldehyde using ethanol as the solvent and basic conditions (50% KOH v/ v) at room temperature for 24 h. The 3,4,5-trimethoxyphenyl chalcone was obtained by vacuum filtration and recrystallized in ethyl acetate. The chalcone was refluxed with hydrazine hydrate and acetic acid as the solvent for 6 h. The solution was then neutralized with sodium bicarbonate solution. The H3TM04 was obtained by vacuum filtration and recrystallized in ethyl acetate. The structure was identified based on the melting point, high-resolution mass spectrometry (HRMS), 1H and 13C nuclear magnetic resonance (NMR) spectroscopy and elementary analysis, as previously described by our group [23]. 2.1.2. Synthesis and characterization of CHC The CHC was obtained using the methodology previously reported by our group [23], which consists of two procedures: i) the insertion of carboxymethyl groups and ii) the insertion of hexanoyl groups. Firstly, the chitosan was suspended in propanol (50 mL), NaOH solution (40% v/v) and chloroacetic acid (25 g) for 48 h to obtain the N,O-carboxymethyl chitosan derivative. Then the product (2 g) was dissolved in water followed by the addition of methanol (50 mL) and hexanoyl anhydride (0.5 mol L−1); the solution was kept under overnight stirring. The CHQ was purified by dialysis against an ethanol solution (50% v/ v), using a dialysis membrane (molecular weight cut off 3.5 kD), for 72 h, followed by the lyophilization of the polymer: the solution was frozen with liquid nitrogen and then submitted to a very low pressure regime, where the solvent was sublimated and only the CHQ was left in the vial. It was then characterized by 1H NMR spectroscopy and Fourier transformed infrared spectroscopy FTIR. The degree hexanoyl substitution (51.60%) and the total fraction of carboxymethyl groups (0.96) were measured in a previous study [22].

A

2.2. Formation of nanoparticles Nanocapsules with (CHC-SDS-H) and without (CHC-SDS) pyrazoline were formed by the following method: 1 mg mL−1 CHC was suspended in 6 mmol L−1 SDS(aq) previously prepared in buffer phosphate pH 7.4 and kept under stirring at 25 °C for 36 h. The sample of CHC-SDS was subjected to ultrasound using a probe-type sonication procedure (UP200S, Hielsher) at 15 W for 20 min to obtain the CHC-SDS nanocapsules. The sonication was conducted in a cold water bath to inhibit the evaporation of the solution during the process. The CHC-SDS-H nanocapsules were obtained by the same method, but with the drop-

B

Fig. 1. Structures of a) carboxymethyl-hexanoyl-chitosan (CHC) and b) pyrazoline (H3TM04). 2

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wise addition of 360 μL of a 10 mmol L−1 H3TM04 acetonitrile solution during the sonication. These particles were then stored in the dark at 4 °C [11].

(75 rpm). The release medium (200,0 mL) used was phosphate buffer solution pH 7.4 with 0.5% (w/v) sodium dodecyl sulfate to attain sink conditions. Suspensions of H3TM04 or H3TM04-loaded nanocapsules (5 mL of sample with 1200 μmol L−1 H3TM04) at pH 7.4 were placed in the dialysis bag. Aliquots (5 mL) of the release medium were removed at different intervals over a 72-h period and the H3TM04 concentration was quantified by fluoresce spectroscopy, based on a calibration curve obtained with an external standard in SDS 0.5% (w/v). The release medium was immediately replaced with fresh medium. The experiments were carried out in triplicate. To evaluate the release kinetics and the mechanism of release, four kinetic models were used to fit the drug release data: the zero-order model, the first-order model, the Higuchi model and the power law model, which can be represented by the following equations (Eqs. (3) to (6)):

2.3. Characterization of CHC-SDS nanoparticles The size distributions of the CHC-SDS-H and CHC-SDS nanoparticles were measured by dynamic light-scattering (DLS) (ALV laser goniometer) with a 35 mW red helium‑neon linearly polarized laser (λ = 632.8 nm) and multiple tau digital correlator (LSE-5004). The samples were previously filtered through a cellulose membrane filter (0.45 μm, Chromafil) and analyzed at scattering angles from 30 to 145° and 298.15 K. ALV-correlator software V.3.0 was used to obtain the DLS auto-correlation functions g(1) (q,t). The distribution function of the decay time A(t) and the distribution function of size A(RH) were obtained using CONTIN analysis of the g(1) (q,t) function [24]. The hydrodynamic radii of the nanocapsules (RHNp) were calculated using the Einstein−Stokes equation (Eq. (1)) [25]: Np RH =

6

kT DNp)

First

order: Mt / M = 1

(3)

e(

k1 t )

(4)

Higuchi model:Mt / M = kH t 2

(5)

Power law model: Mt / M = kt n

(6)

where Mt/M∞ is the fraction of therapeutic drug released at time t and k0, k1, kH and k are the zero-order release constant, first-order release constant, Higuchi constant and power law constant, respectively. The release constants and parameters of the models were calculated by fitting the experimental release data. 2.6. Anti-tumor effects assay 2.6.1. Cell culture An acute myeloid leukemia (K-562) and a T-cell acute lymphoblastic leukemia (Jurkat), both kindly provided by Prof. Dr. Alberto Orfao from Centro de Investigación del Cáncer of the Universidad de Salamanca, were used in this study. The cells were cultured in Roswell Park Memorial Institute (RPMI) medium (GIBCO®, Brazil) supplemented with 10% heat inactivated fetal bovine serum (FBS) (GIBCO®, Brazil), 100 U/mL−1 penicillin (GIBCO®, Brazil) and 10 mmol L−1 HEPES (GIBCO®, Brazil) under 5% CO2 humidified atmosphere, at 37 °C in 25 cm2 and 75 cm2 culture flasks. Cell viability was assessed by the Trypan blue (0.5 w/v) (Sigma-Aldrich®, EUA) exclusion assay and only samples with > 95% of viable cells were used in the experiments.

2.4. Encapsulation efficiency The CHC-SDS-H was formed according to the methodology described in section 2.2 and the non-encapsulated H3TM04 could be removed by centrifugation at 1000 rpm for 5 min, since it is insoluble in water. CHC-SDS-H nanocapsules were then isolated using a higher centrifugation speed (15,000 rpm), followed by suspension in acid solution (pH 4.5) under stirring for 3 h. The resulting suspension was gently mixed with acetonitrile. The concentration of H3TM04 was determined based on a calibration curve (linear range between 0.39 μg mL −1 and 0.27 μg mL −1 of H3TM04) obtained with an external standard by fluorescence spectroscopy (Hitachi F4500). Where the limit of detection and limit of quantification of the fluorescence methods were 0.0066 μg mL−1 and 0.0221 μg mL−1, respectively. The slit width settings of both the excitation and emission monochromators were adjusted to 5 nm. The sample was excited at 300 nm and the fluorescence emission spectra recorded from 320.0 to 520.0 nm. The measurements were performed in triplicate. The total amount of H3TM04 was measured using the same methodology. The percentage of H3TM04 encapsulated (encapsulation efficiency; %EE) was calculated from the following equation (Eq. (2)):

amount of H 3TM 04 encasulated × 100 Total amount of H 3TM 04

order: Mt / M = k 0 t

1

(1)

The morphology of the nanocapsules was analyzed by transmission electron microscopy (TEM 100 kV, JEM-1011). The sample was dropped onto a carbon film-coated copper grid 300 and the excess solution was removed with the tip of a filter paper. A solution of uranyl acetate (0.1%, w/v) was used as a negative staining agent. The sample in the grid was air-dried and analyzed by TEM (100 kV), where the distribution size was determined using the software ImageJ. Zeta potential measurements were performed on a Malvern Zetasizer ZS analyzer (Malvern Instruments) with a capillary electrophoresis cell at 298.15 K. All measurements were carried out in triplicate.

%EE =

Zero

2.6.2. Viability assay (MTT assay) and morphological assessment of apoptosis For the screening, Jurkat cells (5 × 104 cells/well) were incubated for 24 h at different concentrations of SDS, CHC-SDS, and CHC-SDS-H. Cell viability was assessed by the MTT assay (Sigma Chemical Co., USA). In relation to the optical density, the control groups (untreated cells) were considered as 100% of viable cells. To obtain the concentration/time-response curve, the K-562 and Jurkat cells (5 × 104 cells/well) were incubated with different concentrations (5–50 μmol L−1) of H3TM04 encapsulated in the nanocapsules for 24, 48 and 72 h. A maximum volume of 20 μL of the test compounds was added to cells and the same volume of the solvent was added to control wells. Cell viability was assessed by the MTT assay and the IC50 values were calculated using the GraphPad Prism 5 software. The apoptotic death was verified by fluorescence microscope (Leica DM5500 B). K562 and Jurkat cells (5 × 104 cells/well) were incubated with and without CHC-SDS-H nanocapsules in the IC50 for 24 h. The coverslips covering the bottom of the plate were removed, washed with PBS and treated with 40 μL of acridine orange (10 mg mL−1) and ethidium bromide (5 mg mL −1) solution. The cells were analyzed by fluorescence microscopy, where Viable cells exhibited green fluorescence (acridine orange staining) whereas apoptotic cells exhibited an orangered nuclear fluorescence (ethidium bromide staining).

(2)

The %EE was corroborated by the quantification of the total H3TM04 content in the nanocapsule and in the supernatant after ultrafiltration/centrifugation of the nanoparticle, using an Amicon centrifugal filter device with an Ultracel-100 membrane (100 kDa cut-off, Millipore Corp.). 2.5. Drug release kinetics - in vitro study The release studies were performed using the dialysis bag method in a USP Dissolution Apparatus 2 at 37 ± 1 °C with mechanical stirring 3

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(a)

1

1.0

or A(t)

or A(t)

(q,t) (1)

0.4

0.6 0.4

g

(1)

(q,t)

0.6

0.2

0.2 2

-3

2

0.0

0.0 -2

-1

0

1

2

3

-3

-2

-1

log t / (ms) 2

1.0

4

0

1

2

3

log t / (ms)

(c) CHC-SDS CHC-SDS-H

0.8 A(RH)

Fig. 2. Auto-correlation function g(1) (q,t) (1) and DLS distribution function of the decay time (2) of the dynamic light scattering measured at 90o scattering angle for CHC-SDS (a) and CHC-SDS-H (b) and the respective hydrodynamic radius distribution for both curves (c) with mean values of (1) 27.9 nm, 2) 93.5 nm), (3) 11.4 nm and 4) 118.1 nm. The slow mode of the diffusion coefficient represents 98% of the population in both samples.

(b)

0.8

0.8

g

1

1.0

0.6 0.4 1

0.2

3

0.0 0

50

100

150

200

250

300

RH (nm)

the function Γ vs q2 passes through the origin indicates that the relaxation is mainly due to the translational diffusion of the nanocapsules [25]. The diffusion coefficients (DNp) were calculated from the slope of the linear fit from Γ vs q2 curves and the values obtained were 3.27 × 10−12 m s−2 and 2.79 × 10−12 m s−2 for the CHC-SDS and CHC-SDS-H nanocapsules, respectively. From these DNp values, the RHNp can be calculated using the Einstein-Stokes equation (Eq. (1)). The RHNp value for the nanocapsules is larger for CHC-SDS-H (83.9 ± 1.2 nm) compared with CHC-SDS (71.8 ± 0.6 nm). These results are very promising, since only nanoparticles smaller than 200 nm can accumulate in tumor tissue and cross cell membranes [21,26]. The morphology of the nanocapsules was observed by TEM. Fig. 4a shows the transmission electron microscopy images for CHC-SDS, where objects with roughly spherical morphology can be observed (Fig. 4b). The average radius of the nanocapsules measured by TEM (≈35 nm) is lower than the RHNp value measured by DLS (71.8 nm). This difference is probably due to the fact that for the DLS analysis the nanocapsules were swollen with solvent whereas they were completely dry in the TEM measurement. The size of these nanoparticles is in agreement with similar systems already reported in the literature [19,25]. For example, Kun-Ho Liu and co-workers reported the formation of nanocapsules of carboxymethyl hexanoyl chitosan with two size distributions (23.5 ± 0.7 nm and 222.7 ± 2.0 nm) [11]. In order to determine the mean charge of the nanocapsule surface, zeta potential measurements were performed. In the absence of H3TM04, the zeta potential was −30.8 ± 0.1 mV, probably due to the presence of deprotonated carboxymethyl groups from the CHC and sulfate groups from the SDS on the nanoparticle surface. The zeta potential of the CHC-SDS-H was −29.8 ± 0.7 mV, indicating that pyrazoline does not affect the nanoparticle charge. Therefore, we can assume that the H3TM04 is indeed located inside the nanocapsules and thus is not exposed to the interface. The zeta potential measured for both samples reflects the good electrostatic stabilization of these colloidal dispersions [7]. These values are in agreement with those

3. Results and discussion 3.1. Characterization of nanocapsules Nanoparticle size plays a key role in the circulation time, disposition at the tissues from the circulation, tissue distribution, and the endocytose mechanism [12,26]. Thus, the CHC-SDS and CHC-SDS-H nanocapsules were investigated by DLS to obtain the average size. Fig. 2a and b show the correlation function g(1) (q,t) and the distribution function of the decay time A(t) obtained by the Contin method at 90° and 298.15 K. A bimodal distribution could be observed with fast and slow relaxation modes for both samples; the fast mode is probably due to the diffusion of single chains or small aggregates of the amphiphilic chitosan. Since the aggregation process is a dynamic equilibrium, single chains will always co-exist with the nanocapsules, even if we try to remove by using techniques such as ultracentrifugation. This population represents < 2% of the number of total scatterers and was not observed in other techniques. The slow relaxation mode, however, is due to the diffusion of CHC-SDS-H than CHC-SDS nanocapsules. The slower relation time and narrower peak for the CHC-SDS-H sample suggests that the encapsulation of H3TM04 produced larger nanocapsules with a more homogeneous size distribution, as can be observed in Fig. 2c. The apparent hydrodynamic radius of the nanocapsules (RHNp app) at 90° (slow mode) is larger for CHC-SDS-H (118.1 nm) than CHC-SDS (93.5 nm), demonstrating that the encapsulation of pyrazoline increases the average size distribution. These results suggest that the H3TM04 is encapsulated within the core and/or adsorbed at hydrophobic sites on the nanocapsule surface. To determine the real hydrodynamic radius of the nanocapsules (RHNp), DLS measurements were performed at different scattering angles between 30° and 140° for both samples (Fig. 1.A, supplementary material). From the CONTIN analysis of each correlation function, the relaxation times τ were obtained. The angular dependency of the relation frequency Γ (Γ = τ−1) was measured for the CHC-SDS and CHC-SDS-H nanocapsules (Fig. 3a and b). The fact that 4

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2.5

(a)

2.0

2.0

1.5

1.5

-1

/ 1 0 0 0 * (s )

-1

/ 1000* (s )

2.5

1.0 0.5

Fig. 3. Angular dependency of the relation frequency Γ for the slow modes of (a) CHC-SDS and (b) CHC-SDS-H nanocapsules with the pyrazoline model in buffer pH 7.4 at different scattering angles (30° to 140°) and 298.15 K. Red lines correspond to linear fits with intercept at the origin and the correlation coefficients were > 0.998. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(b)

1.0 0.5

0.0

0.0 0

2

4 2

14

6 2

q / 10 * (m )

8

0

2

4 2

14

6

8

2

q / 10 * (m )

obtained by Kun-Ho Liu and co-workers (i.e., −42 to −23 mV) for nanocapsules of CHC with different degrees of hexanoyl substitution [11]. Besides the higher kinetic stability, nanoparticles with negativelycharged surfaces have a higher circulation time and intra-tumoral transport in comparison with nanoparticles with a cationic surface, increasing their chances of entering the tumor tissue [7,27]. The kinetic stability of the CHC-SDS and CHC-SDS-H dispersions was investigated by following the evolution of the size distribution during the shelf life. It was found that for both samples there was no significant change in the size distribution over 90 days (Fig. 3.A, supplementary material).

3.3. Location of the H3TM04 In order to determine the polarity of the microheterogeneous environment to which the H3TM04 is exposed in the nanocapsules, the fluorescent emission spectra for H3TM04 in CHC (aq) and CHC-SDS nanocapsules were obtained. This method allows the location of the drug binding site inside the nanoaggregates to be identified, based on the emission profile in water/dioxane mixtures of known polarity [30–32]. Fig. 5a shows the fluorescence emission spectra for H3TM04 in several water/dioxane mixtures with 10 to 90% of dioxane. The fluorescent intensity increases with the dioxane fraction in the mixture, with a blue shift of the maximum wavelength of emission fluorescence (λEMMax) due to the decreasing polarity of the microheterogeneous environment around the H3TM04 compounds. The λEMMax was correlated with the polarity equivalent parameter of the water-dioxane mixtures ET(30) (Fig. 5b), previously reported by Kosower and co-workers [33]. Assuming that this behavior can be applied to nanoaggregates, the micropolarity environment of H3TM04 inside the CHC and CHC-SDS nanocapsules was determined by the interpolation of the λEMMax values. The ET(30) values calculated for H3TM04 in CHC and CHC-SDS were 57.5 and 54.7 kcal mol−1, respectively. Considering that the ET(30) values for water and dioxane are 61.1 kcal mol−1 and 36.1 kcal mol−1, respectively, the H3TM04 is in a less polar microenvironment in the CHC-SDS nanocapsules compared with the CHC aggregates. Moreover, these results demonstrate that H3TM04 is located inside the nanocapsules, indicating that dodecyl chains increase the hydrophobic niches of the nanocapsule.

3.2. Encapsulation efficiency (EE) The encapsulation efficiency was determined by emission fluorescence spectroscopy using an external standard calibration curve (Fig. 2.A, supplementary material). The encapsulation efficiency using 1200 μmol L−1 of H3TM04 was 96.8% ± 0.1 (293.6 μg mL−1), indicating that the CHC-SDS nanocapsules provide high efficiency for the encapsulation of hydrophobic compounds such as the pyrazoline used in this study. Kun-Ho Liu and co-workers encapsulated doxorubicin in CHC nanocapsules and noted that with an increase in the hydrophobic substitution of hexanoyl groups from 0 to 48% the %EE increased from 26.3 and 46.8% [11]. Therefore, the high %EE values obtained in this study can be attributed to the hydrophobic microheterogeneous environment formed by the interaction between the hexanoyl groups on the chitosan backbone and the dodecyl chains of the SDS surfactant [28,29]. To confirm this hypothesis, we carried out experiments to determine the location of the H3TM04 in the nanocapsules.

3.4. Drug release - in vitro study In vitro drug release and kinetics studies were performed to determine the ability of this new formulation to modulate the drug release

Fig. 4. (a) Transmission electron microscopy (TEM) micrographs of CHC-SDS nanocapsule negatively stained with uranyl acetate. Inset shows a spherical nanoparticle in the micrograph with higher magnification. (b) Size distribution of CHC-SDS nanocapsules (70.1 ± 2.3 nm). 5

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7

(a)

400

6

EM

= 2.86 ET (kcal/mol) + 225.67 nm

(b)

R = 0.993

(x)

5

390 / (nm)

3

4

380

M ax

(i)

3

EM

Fluorescence intensity / * 10

Max

2 1

370 ONCHC ONCHC-SDS

360

0 320

360

400

440

480

520

45

/ (nm)

50

Cumulative released H3tm04 / (%)

55

60

65

-1 ET(30) / (kcal mol )

The release profile obtained in this study indicates that CHC-SDS nanocapsules can be used for the controlled delivery of H3TM04 or other hydrophobic therapeutic drugs with a similar structure. Controlled release is a very important factor in the case of drugs against cancer, because a rapid release could promote low distribution of the drug in the tumor tissue and systemic drug exposure [27]. This situation is often the cause of chemotherapy failure, since the drug concentrations can fluctuate widely between the sub-therapeutic drug level and the toxic drug level during the specified time of drug administration, making it necessary to supply the patient with the drug continually. In addition, side effects caused by the free drug, such as bone marrow cell death, can prohibit the completion of the treatment by the patient. In contrast, in controlled-release delivery, the drug concentration does not present these fluctuations and has been found in concentrations that produce beneficial effects, decreasing the quantity of the drug supplied, reducing the side effects and allowing the patient to finish the treatment [37]. The drug release can occur through several mechanism, such as water diffusion into the polymer matrix, polymer swelling, drug dissolution, and polymer dissolution, degradation and or erosion [38,39]. In order to determine the kinetics associated with the H3TM04 release mechanism, the zero-order model, first-order model, Higuchi model and power law model were applied to the drug release data [40–42]. Table 1 shows the release constants, release exponent (n) and regression coefficients (R2) for the models applied to the data. The mechanism involved in the H3TM04 release from the CHC-SDS nanocapsules was governed by the power law model (best fit, with R2 = 0.9901). The apparent release rate constant of the power law and the release exponent (n) were 1.44 × 10−5 h−1 and 1.327, respectively. The low values for the rate constants confirmed that H3TM04 release from the nanocapsules occurred at slow rate compared with the free drug [42]. The mechanisms associated with the drug release can be inferred from the release exponent (n) value: n = 045 corresponds to a Fickian diffusion mechanism or drug release that is diffusion-controlled; n = 0.45–0.89 corresponds to non-Fickian transport (anomalous diffusion) or drug release that is both diffusion-controlled and erosioncontrolled; n = 0.89 relates to a case II mechanism (polymer relaxation

120 100 80 60 40 20 0 0

Fig. 5. (a) Emission fluorescence spectrum of H3TM04 for different water–dioxane volume fractions (spectra (i) to (x) correspond to % dioxane (v/v) 10, 20, 30, 40, 50, 60, 70, 80 and 90. (b) Maximum emission wavelength versus transition energy ET (30) of H3TM04 in several water–dioxane proportions (10 to 90% of dioxane). The red line corresponds to the linear fit. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

10 20 30 40 50 60 70 80 Time / (h)

Fig. 6. In vitro release of H3TM04 from (●) CHC-SDS nanocapsules and (●) free H3TM04 in aqueous solution in buffer pH 7.4 with 0.5% (w/v) sodium dodecyl sulfate to attain sink conditions. Results are shown as mean ± SD of triplicate points and the solid lines serve as a visual guide only.

[34,35]. Fig. 6 shows the release profile for the H3TM04 encapsulated in the CHC-SDS nanocapsules and the free H3TM04 in acetonitrile over a 72-h period. In the first 5 h, 98.3 ± 0.1% of the free H3TM04 was released and only 2.4 ± 0.2% of the H3TM04 was released from the nanocapsules, verifying the controlled release from the nanocapsule. The H3TM04 release from the nanocapsules increased up to 24.9 ± 3.1% after 24 h. The release then continued to gradually increase reaching 86.6 ± 11.3% after 72 h. Based on reports in the literature, controlled delivery is a characteristic of carboxymethyl derivates. Enhui Zhang and co-workers, for instance, reported a release profile over around 72 h for docetaxel-loaded carboxymethyl derivate nanoparticles [36]. Similar results were reported for doxorubicin release from CHC nanocapsules, where approximately 60% of DOX was released over 72 h.

Table 1 Release kinetics parameters for H3TM04 release from CHC-SDS nanocapsules in aqueous solution with 0.5% (w/v) sodium dodecyl sulfate to attain sink conditions. Mathematical models for H3TM04 release kinetics Zero-order k0 (10x−2 h−1 ) 1.224

First-order R

2

0.9893

−2

k1 (10x

Higuchi h

5.624

−1

)

R

2

kH (10x−2 h−1 ) 1.06

0.8405

Power law R

2

0.8652

k (10x−5 h−1 ) 1.440

R2

n

0.9901

1.327

k0, k1, kH and k are the apparent release rate constants of the respective mathematical models, and n is the release exponent of the power law model. 6

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The results show that SDS at a concentration of 60 μmol L−1 is cytotoxic to Jurkat cells, since it significantly reduced the cell viability when compared to the control group (*P ≥ 0.05, Fig. 8). However, the CHC nanoaggregates and CHC-SDS nanocapsules were not cytotoxic toward the Jurkat cells at the concentrations tested, since no notable effect on the cell viability was observed and there was no significant difference from the control group. Thus, CHC-SDS nanocapsules are biocompatible with Jurkat cells and could be applied in biological assays. Calejo and co-workers [52], demonstrated that the toxicity of surfactants of lysine toward HeLa cells is reduced in the presence of ethyl(hydroxyethyl) cellulose due to the favorable polymer–surfactant interaction leading to the formation of mixed micelles. Thus, the surfactant monomer is less available to interact with the cell membrane, lowering the toxicity [52]. We showed that the CHC-SDS nanocapsules with 60 μmol L−1 SDS are not toxic toward Jurkat cells. Based on the interpretation described by Calejo, it appears that due to the strong SDS–CHC interaction, the SDS molecules are less available for interaction with the cell membrane, reducing the occurrence of cell death. The favorable SDS–CHC interaction was reported by Chamorro et al. [22], who showed that hydrophobic interaction promotes the aggregation, decreasing the hydrodynamic radius and increasing the stability of the nanoaggregates in aqueous solution. Thus, the CHC-SDS nanocapsules can be applied to control the H3TM04 delivery to leukemic cells in the concentration range tested. To evaluate the efficiency of the delivery of H3TM04 encapsulated in the CHC-SDS nanocapsules, MTT assays were carried out on two types of leukemia cell lines: Jurkat and K-562. The cells were incubated with the CHC-SDS nanocapsules in different concentrations of H3TM04 (5 to 50 μmol L−1) for 24, 48 and 72 h (Fig. 8a and b). The CHC-SDS-H nanocapsules induced a cytotoxicity effect on both the K-562 and Jurkat leukemia cell lines in a concentration and time-dependent manner. These results allowed the IC50 values reported in Table 2 to be calculated and compared with those for free H3TM04 previously reported by Stefanes et al [23]. The IC50 results for H3TM04 in free form and in the CHC-SDS-H nanocapsules against both cells tested are similar for 24, 48 and 72 h of incubation, except for the Jurkat cells after 24 h and K-562 cells after 72 h of incubation. In the case of the Jurkat cells, the IC50 for the CHCSDS-H nanocapsules is lower (19.32 ± 1.01 μmol L−1) compared with the free compound (32.05 ± 1.06 μmol L−1), indicating that the CHCSDS-H nanocapsules increase the cytotoxicity toward Jurkat cells. In addition, considering that < 30% of the H3TM04 was released from the nanocapsules at pH 7.4 over 24 h (Fig. 6), the cytotoxicity of H3TM04 is higher when encapsulated. It is important to note that the greatest difference in the IC50 values for the free compound (15.65 ± 0.51 μmol L−1) and the encapsulated compound (12.26 ± 0.69 μmol L−1) was observed for K-562 cells after 72 h of incubation. H3TM04-loaded CHC-SDS nanocapsules IC50 values on acute leukemia cells are in agreement with other studies in the literature reporting the cytotoxic activity of pyrazoline derivatives on hematologic neoplastic cells. Zsoldos-Mády et al. [53], evaluated the cytotoxic effect of 17 glycosidic derivatives of ferrocenyl chalcones and ferrocenyl pyrazolines on acute promyelocytic leukemia cells (HL-60) and found 24 h IC50 values between 1.7 and > 200 μmol L−1. In another study, conducted by Shamsuzzaman et al. [54], the effect of 18 steroidal pyrazoline derivatives on Jurkat cells was evaluated after 48 h of incubation and found IC50 values between 10.6 μmol L−1 and 46.3 μmol L−1. The difference in the effect of H3TM04-loaded CHC-SDS nanocapsules on Jurkat and K-562 cells can be explained by the particularities of each type of leukemia cells. K-562 cells were established from a 53-year-old patient diagnosed with chronic myeloid leukemia in blast crisis, which is considered to be an aggressive disease and with unfavorable prognosis. In addition, the oncogenic protein BCR-ABL is expressed in this cell line, which is a characteristic of resistance to treatments [51,54] Thus, the K562 cell line is more resistant and

or swelling-controlled); and n > 0.89 relates to a super case II mechanism (transport or drug release that is erosion-controlled) [41,42]. Therefore, according to the release exponent results, the H3TM04 release from the CHC-SDS nanocapsules occurs via an erosion-controlled mechanism. According to the power law model, in the erosion-controlled mechanism, the release of the compound is driven by two main factors: i) anomalous non-Fickian diffusion (i.e., a diffusion not directly proportional to the concentration gradient) and ii) the nanocapsules erosion. The anomalous diffusion dependents on several factors, such as degree of swelling, dissolution of polymer chains and morphological changes in the polymeric matrix [21,38]. At the same time, as enough water enters into the aggregate, the water-polymer interactions become more possible than polymer-polymer interactions, leading to the separation of polymer chains and the slow disassembly of the nanoparticle. This delivery mechanism has been reported for other biopolymer systems, and it is appropriate for controlled drug delivery due to the protection of the drug from the microenvironment outside the nanoparticle [35,38,43,44]. 3.5. Cytotoxic effect of H3TM04-loaded CHC-SDS nanocapsules on acute leukemia cell lines Due to the severe adverse effects related to the chemotherapy treatment currently used for malignant neoplasms, it is necessary to investigate new compounds with cytotoxic activity on leukemic cells with little or no effect on healthy cells [23,45–47]. Despite its extensive application, SDS is considered toxic and this limits its use in the pharmaceutical and biology fields [48]. Cytotoxicity assays are widely applied to identify possible applications for nanoparticles in biology systems [1,36,45,49]. The Jurkat cell line was established from the peripheral blood of a 14-year-old patient diagnosed with T-ALL relapse [50]. It is known that, in general, younger patients with ALL have a better prognosis, with a high chance of cure [51]. Since these cells are not very resistant, they were selected to evaluate the cytotoxicity of the CHC nanoaggregates, CHC-SDS nanocapsules and free SDS without the presence of H3TM04. Fig. 7 shows the cell viability of Jurkat cells exposed to different concentrations of CHC (0 to 50 μg mL−1) and SDS (0 to 60 μmol L−1) for 24 h of incubation. -1

CSDS / ( mol L )

0

0.7

6.6

16.5

33

60

120 Np

Np-SDS

SDS

Viability / (%)

90

*

60 30 0 0

0.1

1

2.5-1

5

50

CNC / ( g mL ) Fig. 7. Cytotoxity of CHC aggregates, CHC-SDS nanocapsules and SDS toward Jurkat cells. Jurkat cells (5 × 104 cells/well) were incubated with the nanoparticles (0 to 50 μg mL-1) and/or SDS (0 to 60 μmol L-1) for 24 h. The cell viability was evaluated through MTT assay. Optical density of the control groups was considered as 100% cell viability. The results are mean ± MSE of at least 4 independent experiments. ⁎P ≥ 0.05 compared to control groups, using ANOVA followed by the Bonferroni post-hoc test. 7

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120

Viability / (%)

90

48 h

72 h

120

(a)

24 h

48 h *

90

** **

**

Viability / (%)

24 h

*** ***

60

72 h

(b)

*** ***

60

30

30 ***

***

0

0 0

5

10

25

0

50

10

25

50

CH3tm04 / (µmol L )

CH3tm04 / (µmol L )

(c)

5

-1

-1

K-562 Cells Acridine Orange

overlay

CHC-SDS- H3TM04

Controle

Ethidium Bromide

CHC-SDS- H3TM04

Controle

Jurkat Cells

Fig. 8. Cytotoxity of H3TM04-loaded CHC-SDS nanocapsules toward two types of leukemia cell lines: a) Jurkat and b) K-562. Jurkat and K-562 Cells (5 × 104 cells/ well) were incubated with the nanocapsules at different concentrations of H3TM04 (5 to 50 μmol L−1) for 24, 48 and 72 h. The cell viability was evaluated through MTT assays. Optical density of the control groups was considered as 100% cell viability. The results are mean ± MSE of at least 4 independent experiments. ⁎ P ≥ 0.05 ⁎⁎P ≥ 0.01 ⁎⁎⁎P ≥ 0.001* compared to control groups, using ANOVA followed by the Bonferroni post-hoc test. c) Detection of apoptosis on K-562 and Jurkat cells using the acridine orange/ethidium bromide staining assay. Viable cells exhibited green fluorescence (acridine orange staining) whereas apoptotic cells exhibited orange-red nuclear fluorescence (ethidium bromide staining). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

8

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Appendix A. Supplementary data

Table 2 IC50 values for H3TM04 encapsulated in CHC-SDS nanocapsules against K-562 and Jurkat cell lines after incubation for 24, 48 and 72 h. Incubation time (h) 24 48 72 a

IC50 (μmol L−1)a Jurkat

K-562

32.05 ± 1.06 16.19 ± 0.32 15.13 ± 1.05

30.77 ± 0.59 17.22 ± 0.60 15.65 ± 0.51

IC50 – nanocapsules (μmol L−1)

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.msec.2019.110051.

Jurkat

References

19.32 ± 1.01 15.53 ± 0.57 15.60 ± 0.98

K-562 31.78 ± 2.64 19.91 ± 1.45 12.26 ± 0.69

[1] H. Wang, P. Agarwal, S. Zhao, R.X. Xu, J. Yu, X. Lu, X. He, Hyaluronic acid-decorated dual responsive nanoparticles of Pluronic F127, PLGA, and chitosan for targeted co-delivery of doxorubicin and irinotecan to eliminate cancer stem-like cells, Biomaterials 72 (2015) 74–89, https://doi.org/10.1016/j.biomaterials.2015.08. 048. [2] Q. Tian, C.N. Zhang, X.H. Wang, W. Wang, W. Huang, R.T. Cha, C.H. Wang, Z. Yuan, M. Liu, H.Y. Wan, H. Tang, Glycyrrhetinic acid-modified chitosan/poly (ethylene glycol) nanoparticles for liver-targeted delivery, Biomaterials 31 (2010) 4748–4756, https://doi.org/10.1016/j.biomaterials.2010.02.042. [3] Y. Bian, D. Gao, Y. Liu, N. Li, X. Zhang, R.Y. Zheng, Q. Wang, L. Luo, K. Dai, Preparation and study on anti-tumor effect of chitosan-coated oleanolic acid liposomes, RSC Adv. 5 (2015) 18725–18732, https://doi.org/10.1039/c4ra13860k. [4] K. Chatterjee, J. Zhang, N. Honbo, J.S. Karliner, Doxorubicin cardiomyopathy, Cardiology 115 (2010) 155–162, https://doi.org/10.1159/000265166. [5] G. Chen, I. Roy, C. Yang, P.N. Prasad, Nanochemistry and nanomedicine for nanoparticle-based diagnostics and therapy, Chem. Rev. 116 (2016) 2826–2885, https://doi.org/10.1021/acs.chemrev.5b00148. [6] M. Mohammadi, M. Ramezani, K. Abnous, M. Alibolandi, Biocompatible polymersomes-based cancer theranostics: towards multifunctional nanomedicine, Int. J. Pharm. 519 (2017) 287–303, https://doi.org/10.1016/j.ijpharm.2017.01.037. [7] V.R. Devadasu, V. Bhardwaj, M.N.V.R. Kumar, Can controversial nanotechnology promise drug delivery? Chem. Rev. 113 (2013) 1696–1735, https://doi.org/10. 1021/cr300047q. [8] A. Kumari, S.K. Yadav, S.C. Yadav, Biodegradable polymeric nanoparticles based drug delivery systems, Colloids Surf. B: Biointerfaces 75 (2010) 1–18, https://doi. org/10.1016/j.colsurfb.2009.09.001. [9] P. Kothamasu, H. Kanumur, N. Ravur, C. Maddu, R. Parasuramrajam, S. Thangavel, Nanocapsules: the weapons for novel drug delivery systems, BioImpacts 2 (2012) 71–81, https://doi.org/10.5681/bi.2012.011. [10] E. Kim, D. Kim, H. Jung, J. Lee, S. Paul, N. Selvapalam, Y. Yang, N. Lim, C.G. Park, K. Kim, Facile, template-free synthesis of stimuli-responsive polymer nanocapsules for targeted drug delivery, Angew. Chem. Int. Ed. 49 (2010) 4405–4408, https:// doi.org/10.1002/anie.201000818. [11] K.H. Liu, S.Y. Chen, D.M. Liu, T.Y. Liu, Self-assembled hollow nanocapsule from amphiphatic carboxymethyl-hexanoyl chitosan as drug carrier, Macromolecules 41 (2008) 6511–6516, https://doi.org/10.1021/ma8002399. [12] N.P. da Silva Santos, S.C. Nascimento, M.S.O. Wanderley, N.T. Pontes-Filho, J.F. da Silva, C.M.M.B. de Castro, E.C. Pereira, N.H. da Silva, N.K. Honda, N.S. SantosMagalhães, Nanoencapsulation of usnic acid: an attempt to improve antitumour activity and reduce hepatotoxicity, Eur. J. Pharm. Biopharm. 64 (2006) 154–160, https://doi.org/10.1016/j.ejpb.2006.05.018. [13] B. Stella, S. Arpicco, F. Rocco, V. Marsaud, J.M. Renoir, L. Cattel, P. Couvreur, Encapsulation of gemcitabine lipophilic derivatives into polycyanoacrylate nanospheres and nanocapsules, Int. J. Pharm. 344 (2007) 71–77, https://doi.org/10. 1016/j.ijpharm.2007.06.006. [14] C.E. Mora-Huertas, H. Fessi, A. Elaissari, Polymer-based nanocapsules for drug delivery, Int. J. Pharm. 385 (2010) 113–142, https://doi.org/10.1016/j.ijpharm. 2009.10.018. [15] H. Yi, L.Q. Wu, W.E. Bentley, R. Ghodssi, G.W. Rubloff, J.N. Culver, G.F. Payne, Biofabrication with chitosan, Biomacromolecules 6 (2005) 2881–2894, https://doi. org/10.1021/bm050410l. [16] J.P. Jee, J.H. Na, S. Lee, S.H. Kim, K. Choi, Y. Yeo, I.C. Kwon, Cancer targeting strategies in nanomedicine: design and application of chitosan nanoparticles, Curr. Opin. Solid State Mater. Sci. 16 (2012) 333–342, https://doi.org/10.1016/j.cossms. 2013.01.002. [17] S.K. Shukla, A.K. Mishra, O.A. Arotiba, B.B. Mamba, Chitosan-based nanomaterials: a state-of-the-art review, Int. J. Biol. Macromol. 59 (2013) 46–58, https://doi.org/ 10.1016/j.ijbiomac.2013.04.043. [18] V. Zargar, M. Asghari, A. Dashti, A review on chitin and chitosan polymers: structure, chemistry, solubility, derivatives, and applications, Chem. Bio. Eng. Rev. 2 (2015) 204–226, https://doi.org/10.1002/cben.201400025. [19] X. Jin, R. Mo, Y. Ding, W. Zheng, C. Zhang, Paclitaxel-loaded N-octyl-O-sulfate chitosan micelles for superior cancer therapeutic efficacy and overcoming drug resistance, Mol. Pharm. 11 (2014) 145–157, https://doi.org/10.1021/mp400340k. [20] M. Huo, Y. Zhang, J. Zhou, A. Zou, J. Li, Formation, microstructure, biodistribution and absence of toxicity of polymeric micelles formed by N-octyl-N,O-carboxymethyl chitosan, Carbohydr. Polym. 83 (2011) 1959–1969, https://doi.org/10.1016/j. carbpol.2010.10.073. [21] Q. Mu, G. Jiang, L. Chen, H. Zhou, D. Fourches, A. Tropsha, B. Yan, Chemical basis of interactions between engineered nanoparticles and biological systems, Chem. Rev. (2016) 7740–7781, https://doi.org/10.1021/cr400295a.Chemical. [22] A.F.C. Rengifo, S.C. Santos, V.R. de Lima, A.J.P. Agudelo, L.H.M. da Silva, A.L. Parize, E. Minatti, Aggregation behavior of self-assembled nanoparticles made from carboxymethyl-hexanoyl chitosan and sodium dodecyl sulphate surfactant in water, J. Mol. Liq. 278 (2019) 253–261. [23] N.M. Stefanes, J. Toigo, M.F. Maioral, A.V. Jacques, L.D. Chiaradia-Delatorre, D.M. Perondi, A.A.B. Ribeiro, Á. Bigolin, I.M.S. Pirath, B.F. Duarte, R.J. Nunes,

IC50 values reported by Stefanes et al [23].

aggressive than the Jurkat cell line, which was established from a patient diagnosed with T-ALL with a better prognosis [50–55]. The capacity of H3TM04-loaded nanocapsules to induce morphological changes on Jurkat and K-562 cells was evaluated by using acridine orange/ethidium bromide staining (Fig. 8c). The control cells, when stained with acridine orange, exhibited a green color and nuclei integrity, indicating the cell viability. The cells treated with H3TM04loaded CHC-SDS nanocapsules for 24 h showed morphological changes when compared with the control cells. In the green cells, the membrane was intact but showed condensation of the chromatin and nuclear fragmentation. The leukemic cells stained with orange acridine (green color) and ethidium bromide (orange/red color) presented decreased of cell volume, condensation of the chromatin, formation of plasma membrane extensions and loss of membrane integrity, suggesting the apoptosis mechanism as demonstrated by Stefanes et al. when using free H3TM04. These results allow to infer that the nanocapsules were indeed delivering H3TM04 to the cells and H3TM04-loaded CHC-SDS nanocapsules may serve as a therapeutic alternative for the treatment of acute leukemias, however further studies are needed to evaluate the effect on healthy cells and the mechanisms involved in cytotoxicity to leukemic cells. 4. Conclusions In this study we developed novel CHC-SDS nanocapsules with spherical morphology composed of CHC and SDS. The SDS increases the hydrophobic domains within the nanocapsules and promotes a high capacity for the encapsulation of the pyrazoline H3TM04 (96.8%). The CHC-SDS-H nanocapsules provide controlled H3TM04 delivery via an erosion-controlled mechanism. The cytotoxicity assays showed that the CHC-SDS nanocapsules are biocompatible and non-toxic. In addition, the encapsulation of H3TM04 into CHC-SDS nanocapsules decreased the IC50 for Jurkat and K-562 leukemia cells after 24 h and 72 h of incubation, respectively. Our results demonstrate that CHC-SDS nanocapsules represent a promising drug delivery system for pyrazoline derivates and hydrophobic drugs. Acknowledgments We gratefully acknowledge for the Brazilian governmental agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financially supporting this project and for student scholarships given to Andrés F. C. Renfigo, Jessica Toigo, Natalia Stefanes and Cassiana Mendes. Santos-Silva MC is recipient of a Research Fellowship from the CNPq (Brazil). The authors would like to thank the LCME-UFSC for technical support during electron microscopy work. Declaration of competing interest The authors declare no conflicting interests. 9

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[24] [25] [26]

[27] [28] [29] [30]

[31]

[32]

[33]

[34]

[35] [36]

[37] [38]

5b00346. [39] R.S. Langer, New methods of drug delivery, Science (1990) 1527–1533, https://doi. org/10.1126/science.2218494. [40] R.S. Tiǧli Aydin, M. Pulat, 5-fluorouracil encapsulated chitosan nanoparticles for pH-stimulated drug delivery: evaluation of controlled release kinetics, J. Nanomater. 2012 (2012) 1–10, https://doi.org/10.1155/2012/313961. [41] R.K. Das, N. Kasoju, U. Bora, Encapsulation of curcumin in alginate-chitosanpluronic composite nanoparticles for delivery to cancer cells, Nanomedicine 6 (2010) 153–160, https://doi.org/10.1016/j.nano.2009.05.009. [42] M.D.A. Muhsin, G. George, K. Beagley, V. Ferro, H. Wang, N. Islam, Effects of chemical conjugation of l-leucine to chitosan on dispersibility and controlled release of drug from a nanoparticulate dry powder inhaler formulation, Mol. Pharm. 13 (2016) 1455–1466, https://doi.org/10.1021/acs.molpharmaceut.5b00859. [43] B. Fonseca-Santos, M. Chorilli, An overview of carboxymethyl derivatives of chitosan: their use as biomaterials and drug delivery systems, Mater. Sci. Eng. C 77 (2017) 1349–1362, https://doi.org/10.1016/j.msec.2017.03.198. [44] A. Göpferich, Mechanisms of polymer degradation and erosion, Biomaterials 17 (1996) 103–114, https://doi.org/10.1016/0142-9612(96)85755-3. [45] C.P. Stewart, B. W., Wild, World Cancer Report 2014, Lyon (2014). [46] J. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D.M. Parkin, D. Forman, F. Bray, Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012, Int. J. Cancer 136 (2015) 66–67, https:// doi.org/10.1002/ijc.29210. [47] Q. Feng, W.S. Leong, L. Liu, W.I. Chan, Peruvoside, a cardiac glycoside, induces primitive myeloid leukemia cell death, Molecules 21 (2016), https://doi.org/10. 3390/molecules21040534. [48] L. Dong, C.M. Witkowski, M.M. Craig, M.M. Greenwade, K.L. Joseph, Cytotoxicity effects of different surfactant molecules conjugated to carbon nanotubes on human astrocytoma cells, Nanoscale Res. Lett. 4 (2009) 1517–1523, https://doi.org/10. 1007/s11671-009-9429-0. [49] Z. Amoozgar, J. Park, Q. Lin, Y. Yeo, Low molecular-weight chitosan as a pH-sensitive stealth coating for tumor-specific drug delivery, Mol. Pharm. 9 (2012) 1262–1270, https://doi.org/10.1021/mp2005615. [50] U. Schneider, H.-U. Schwenk, G. Bornkamm, Characterization of EBV-genome negative “null” and “T” cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed non-Hodgkin lymphoma, Int. J. Cancer 19 (1977) 621–626, https://doi.org/10.1002/ijc.2910190505. [51] J. Swerdlow, S. H.; Campo, E.; Harris, N. L.; Jaffe, E. S.; Pileri, S. A.; Stein, H.; Thiele, WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues (Revised 4th edition)., Lyon, 2017. [52] M.T. Calejo, A.M.S. Cardoso, E.F. Marques, M.J. Araújo, A.L. Kjøniksen, S.A. Sande, M.C.P. de Lima, A.S. Jurado, B. Nyström, In vitro cytotoxicity of a thermoresponsive gel system combining ethyl(hydroxyethyl) cellulose and lysine-based surfactants, Colloids Surf. B: Biointerfaces 102 (2013) 682–686, https://doi.org/10.1016/j. colsurfb.2012.09.033. [53] V. Zsoldos-Mády, A. Csámpai, R. Szabó, E. Mészáros-Alapi, J. Pásztor, F. Hudecz, P. Sohár, Synthesis, structure, and in vitro antitumor activity of some glycoside derivatives of ferrocenyl-chalcones and ferrocenyl-pyrazolines, ChemMedChem 1 (2006) 1119–1125, https://doi.org/10.1002/cmdc.200600052. [54] Shamsuzzaman, H. Khanam, A. Mashrai, A. Sherwani, M. Owais, N. Siddiqui, Synthesis and anti-tumor evaluation of B-ring substituted steroidal pyrazoline derivatives, Steroids 78 (2013) 1263–1272, https://doi.org/10.1016/j.steroids.2013. 09.006. [55] M.F. Maioral, P.C. Gaspar, G.R. Rosa Souza, A. Mascarello, L.D. Chiaradia, M.A. Licínio, A.C.R. Moraes, R.A. Yunes, R.J. Nunes, M.C. Santos-Silva, Apoptotic events induced by synthetic naphthylchalcones in human acute leukemia cell lines, Biochimie 95 (2013) 866–874, https://doi.org/10.1016/j.biochi.2012.12.001.

M.C. Santos-Silva, Synthesis of novel pyrazoline derivatives and the evaluation of death mechanisms involved in their antileukemic activity, Bioorg. Med. Chem. 27 (2019) 375–382, https://doi.org/10.1016/j.bmc.2018.12.012. S.W. Provencher, P. Štěpánek, Global analysis of dynamic light scattering autocorrelation functions, Part. Part. Syst. Charact. 13 (1996) 291–294, https://doi.org/ 10.1002/ppsc.19960130507. E.V. Korchagina, O.E. Philippova, Ion-specific self-assembly of hydrophobically modified polycation of natural origin, Macromolecules 48 (2015) 8622–8628, https://doi.org/10.1021/acs.macromol.5b02213. T. Ramasamy, H.B. Ruttala, B.G. Kanu, B.K. Poudel, H.-G. Choi, C.S. Yong, J.O. Kim, Smart chemistry-based nanosized drug delivery systems for systemic applications: a comprehensive review, J. Control. Release 258 (2017) 226–253, https://doi.org/ 10.1016/j.jconrel.2017.04.043. Z. Wang, Z. Wu, J. Liu, W. Zhang, Particle morphology: an important factor affecting drug delivery by nanocarriers into solid tumors, Expert Opin. Drug Deliv. (2017) 1–17, https://doi.org/10.1080/17425247.2018.1420051. C. Onesippe, S. Lagerge, Study of the complex formation between sodium dodecyl sulfate and hydrophobically modified chitosan, Carbohydr. Polym. 74 (2008) 648–658, https://doi.org/10.1016/j.carbpol.2008.04.021. C. Onesippe, S. Lagerge, Study of the complex formation between sodium dodecyl sulfate and chitosan, Colloids Surf. A Physicochem. Eng. Asp. 317 (2008) 100–108, https://doi.org/10.1016/j.colsurfa.2007.10.002. R. Mondal, N. Ghosh, S. Mukherjee, Enhanced binding of phenosafranin to triblock copolymer F127 induced by sodium dodecyl sulfate: a mixed micellar system as an efficient drug delivery vehicle, J. Phys. Chem. B 120 (2016) 2968–2976, https:// doi.org/10.1021/acs.jpcb.6b00759. S. Ghosh, J. Kuchlyan, D. Banik, N. Kundu, A. Roy, C. Banerjee, N. Sarkar, Organic additive, 5-methylsalicylic acid induces spontaneous structural transformation of aqueous pluronic triblock copolymer solution: a spectroscopic investigation of interaction of curcumin with pluronic micellar and vesicular aggregates, J. Phys. Chem. B 118 (2014) 11437–11448, https://doi.org/10.1021/jp507378w. B.K. Paul, N. Guchhait, Spectroscopic probing of location and dynamics of an environment-sensitive intramolecular charge transfer probe within liposome membranes, J. Colloid Interface Sci. 363 (2011) 529–539, https://doi.org/10.1016/j. jcis.2011.07.074. E.M. Kosower, H. Dodiuk, K. Tanizawa, M. Ottolenghi, N. Orbachlc, Intramolecular donor-acceptor systems. Radiative and nonradiative processes for the excited states of 2-N-arylamino-6-naphthalenesulfonates, J. Am. Chem. Soc. 97 (1975) 2167–2178, https://doi.org/10.1021/ja00841a030. P. Figueiredo, K. Lintinen, A. Kiriazis, V. Hynninen, Z. Liu, T. Bauleth-Ramos, A. Rahikkala, A. Correia, T. Kohout, B. Sarmento, J. Yli-Kauhaluoma, J. Hirvonen, O. Ikkala, M.A. Kostiainen, H.A. Santos, In vitro evaluation of biodegradable ligninbased nanoparticles for drug delivery and enhanced antiproliferation effect in cancer cells, Biomaterials 121 (2017) 97–108, https://doi.org/10.1016/j. biomaterials.2016.12.034. P.T. Wong, S.K. Choi, Mechanisms of drug release in nanotherapeutic delivery systems, Chem. Rev. 115 (2015) 3388–3432, https://doi.org/10.1021/cr5004634. E. Zhang, R. Xing, S. Liu, K. Li, Y. Qin, H. Yu, P. Li, Comparison in docetaxel-loaded nanoparticles based on three different carboxymethyl chitosans, Int. J. Biol. Macromol. 101 (2017) 1012–1018, https://doi.org/10.1016/j.ijbiomac.2017.03. 195. K.E. Uhrich, S.M. Cannizzaro, R.S. Langer, K.M. Shakesheff, Polymeric systems for controlled drug release, Chem. Rev. 99 (1999) 3181–3198, https://doi.org/10. 1021/cr940351u. N. Kamaly, B. Yameen, J. Wu, O.C. Farokhzad, Degradable controlled-release polymers and polymeric nanoparticles: mechanisms of controlling drug release, Chem. Rev. 116 (2016) 2602–2663, https://doi.org/10.1021/acs.chemrev.

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