Synthesis and characterization of non-toxic and thermo-sensitive poly(N-isopropylacrylamide)-grafted cashew gum nanoparticles as a potential epirubicin delivery matrix

Carbohydrate Polymers 154 (2016) 77–85

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Synthesis and characterization of non-toxic and thermo-sensitive poly(N-isopropylacrylamide)-grafted cashew gum nanoparticles as a potential epirubicin delivery matrix Clara M.W.S. Abreu a , Haroldo C.B. Paula b , Vitor Seabra c , Judith P.A. Feitosa a , Bruno Sarmento c,d,e , Regina C.M. de Paula a,∗ a Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, Caixa Postal 6021, Rua Humberto Monte s/n, Campus do Pici, CEP 60455-760 Fortaleza, Brazil b Departamento de Química Analítica e Físico-Química, Universidade Federal do Ceará, Caixa Postal 6021, Rua Humberto Monte s/n, Campus do Pici, CEP 60455-760 Fortaleza, Brazil c CESPU, Instituto de Investigac¸ão e Formac¸ão Avanc¸ada em Ciências e Tecnologias da Saúde and Instituto Universitário de Ciências da Saúde, Rua Central de Gandra 1317, 4585-116 Gandra, Portugal d INEB – Instituto de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen, 208, 4200-135 Porto, Portugal e i3S – Instituto de Investigac¸ão e Inovac¸ão em Saúde, Universidade do Porto, Rua Alfredo Allen, 208, 4200-135 Porto, Portugal

a r t i c l e

i n f o

Article history: Received 2 June 2016 Received in revised form 9 August 2016 Accepted 9 August 2016 Available online 10 August 2016 Keywords: N-isopropylacrylamide Nanoparticles Thermo-sensitive Cytotoxicity Epirubicin

a b s t r a c t Cashew gum (CG) was grafted with N-isopropylacrylamide (NIPA) by radical polymerization to originate a stimuli-sensitive copolymer for drug delivery purposes. NMR and IR spectroscopy confirmed the insertion of NIPA onto the cashew gum chains. The graft copolymer (CG:NIPA) demonstrated thermal responsiveness. The critical aggregation concentration of the copolymers at 25 ◦ C was higher than at 50 ◦ C. At temperatures lower than the LCST, the nanoparticle size ranged from 12 to 21 nm, depending on the CG:NIPA ratio, but above the LCST the particles aggregated, increasing the particle size. Regarding the potential for future oral application, the nanoparticles showed no cytotoxic activity against the Caco2 and HT29-MTX intestine cell lines. Epirubicin was encapsulated into nanoparticles of CG-NIPA (1:1), resulting in a 64% association efficiency and 22% loading capacity. Thus, the CG:NIPA graft copolymer demonstrates good potential for used in controlled drug delivery systems. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction The increasing use of nanoparticles as drug delivery systems has resulted in significant improvements in the pharmacokinetics of therapeutic active molecules. Nanoparticles have been particularly efficient in increasing the solubility and stability of drugs, enhancing the administration safety and improving the biodistribution pattern, leading to more physiologically compatible and less toxic profiles (Luo et al., 2012a). Core-shell type (hydrophilic shell - hydrophobic core) nanopolymer aggregates, in the form of micelles, may be more suitable as drug delivery systems because of their greater structural stability gained from the intertwining of their chains and the intermolecular interactions between hydrophobic segments (Kang, Na, & Bae, 2003). These nanosystems are generally produced from amphiphilic copolymers, which spontaneously organize into

∗ Corresponding author. E-mail address: [email protected] (R.C.M. de Paula). http://dx.doi.org/10.1016/j.carbpol.2016.08.031 0144-8617/© 2016 Elsevier Ltd. All rights reserved.

micelles through intra or intermolecular associations when in contact with an aqueous medium (Bigot et al., 2010). Polysaccharides appear to be appropriate for preparing nanoparticles, especially those for biomedical purpose, due to their unique physicochemical properties and excellent biocompatibility (Sosnik, das Neves & Sarmento, 2014). Moreover, they are very safe and highly biodegradable and there are abundant sources in nature which are associated with low cost processing (Yang, Han, Zheng, Dong, & Liu, 2015). Furthermore, the relatively high molecular weight of polysaccharides means that the release rate of the loads can be controlled and allows biodegradation to occur before rapid renal clearance (Basu, Kunduru, Abtew, & Domb, 2015). The presence of several functional groups, such as hydroxyl, carboxyl, sulfate and amino, allows polysaccharides to be modified through the insertion of hydrophobic or hydrophilic groups. The insertion of a polymer chain or the polymerization of a monomer onto a polysaccharide can promote the formation of self-assembled nanoparticles. These nanoparticles may be responsive to stimuli and drug release can be promoted through a change in the nanoparticle volume in response to variations in the pH, or temperature or

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ionic strength or the presence of a magnetic field (Maya et al., 2013; Nitta & Numata, 2013). In this regard, the use of the polysaccharide obtained from cashew gum shows promise. Cashew gum is a heteropolysaccharide composed of galactose, glucose, arabinose and glucuronic acid (de Paula & Rodrigues, 1995; De Paula, Heatley & Budd, 1998). It presents no toxicity (Quelemes et al., 2013) and low viscosi ty (de Paula & Rodrigues, 1995) and its production is feasible, since its exudation is simple, often spontaneous and provides high yields (Cunha, de Paula, & Feitosa, 2009). In fact, cashew gum offers good potential for commercial production, since the cashew tree (Anacardium occidentale L.) is extensively cultivated in tropical regions of the world. The literature also shows that it has potential for biomedical applications and provides good yields in chemical modifications (Das, Nayak, & Nanda, 2013; Moura Neto, Maciel, Cunha, de Paula, & Feitosa, 2011; Gowthamarajan, Jawahar, Wake, Jain, & Sood, 2012; Quelemes et al., 2013). Cashew nanoparticles have been prepared via different routes, for instance, acrylic acid-grafted-cashew gum has been prepared by radical polymerization using Ce (IV) ions as the initiator (Silva, Feitosa, Paula, & de Paula, 2009), by polyelectrolyte complexation of carboxymethylated cashew gum and chitosan (Silva, Maciel, Feitosa, Paula, & de Paula, 2010) and by the self-assembly of acetylated cashew gum prepared through the dialysis of an organic solution (DMSO) against a non-solvent (water) (Pitombeira et al., 2015). Poly-N-isopropylacrylamide (PNIPA) is a thermoresponsive material with a critical solution temperature (LCST) close to 32 ◦ C. This polymer has therefore been extensively investigated for use in biomedical applications, including drug delivery, mainly in association with biodegradable materials (Chen, Chen, Nan, Wang & Chu, 2012; Lizundia, Meaurio, Laza, Vilas & León Isidro, 2015; Lv et al., 2011; Zhang et al., 2009). Although PNIPA is not biodegradable and may raise toxicity concerns related to its continued residence in the intraocular cavity (Lai & Hsieh, 2012), it is reported that PNIPA particles present no adverse geno- or cytotoxicological effects toward many cell lines (Naha et al., 2010; Rejinold, Sreerekha, Chennazhi, Nair & Jayakumar, 2011), indicating that once formulated into nanoparticles it can be considered as biocompatible. Poly-N-isopropylacrylamide grafting onto another polymer, such as a polysaccharide, is of greater interest than the copolymerization of NIPA with other synthetic polymers, since the continuous stream of the PNIPA chain can be broken, reducing the interaction between isopropyl groups above the LCST (Fundueanu, Constantin & Ascenzi, 2008; Fundueanu et al., 2010). In this context, the aim of this study was to synthesize and characterize thermoresponsive nanoparticles of cashew gum grafted with N-isopropylacrylamide for potential biomedical applications. No reports of cashew gum grafted with N-isopropylacrylamide could be found in the literature and this is the first time that the toxicity of cashew gum and it derivatives against Caco-2 and HT29-MTX cells has been evaluated. As a proof of concept for the application of CG-NIPA nanoparticles as a delivery matrix, the association efficiency and loading capacity of epirubicin (EPI) were evaluated. EPI is an anthracycline and a clinically useful chemotherapy agent, but it shows poor aqueous solubility and severe side effects.

2. Experimental 2.1. Materials Crude samples of the cashew tree (Anacardium occidentale) exudate were provided by Embrapa Agroindústria Tropical, Fortaleza, Ceará, Brazil. The cashew gum (CG) was obtained as a sodium salt

using a previously described method (Rodrigues, de Paula, & Costa, 1993). N-isopropylacrylamide 97% (NIPA) and thiazolyl blue tetrazolium bromide were obtained from Sigma-Aldrich. Cerium (IV) ammonium nitrate (CAN) was purchased from Acrós Organics and epirubicin hydrochloride from Laboratório Químico Farmacêutico Bergamo Ltda (Brazil). All reagents were used as received. Cashew gum used in this work has molar mass, obtained by size exclusion chromatography of 6.9 × 104 g/mol and the molar sugar ratio for galactose:glucose:arabinose:rhamnose:glucuronic acid of 1:0.2:0.08:0.05:0.06 respectively. The NMR of this purified sample is similar to previously published results on cashew gum and is shown in the Supplementary file (de Paula & Rodrigues, 1995; de Paula, Heatley & Budd, 1998).

2.2. Synthesis and purification of graft copolymers The method used for the synthesis of the graft copolymers was adapted from a procedure involving the grafting of acrylic acid onto dextran gum (Tang, Dou, & Sun, 2006). One gram of CG was dissolved in distilled water (50 mL) at room temperature under stirring overnight. After dissolution, nitrogen was bubbled for 30 min and a solution of CAN in 0.1 M nitric acid and a designated amount of NIPA were then added successively. The molar ratio of the NIPA:CAN used in this study was 1:0.07. Three CG:NIPA ratios (1:0.5; 1:1 and 1:2) were used. The CG:NIPA molar ratio was calculated considering the monosaccharide/NIPA units. The reaction was kept for 4 h under an N2 atmosphere at 25 ◦ C. In the next step, 5 M NaOH was added to neutralize the reaction system. The reaction solution was dialyzed against distilled water for 6 days using a membrane bag with a molecular weight cut-off of 12,000 to remove the unreacted monomers and the ungrafted NIPA. The final aqueous solutions were lyophilized to obtain the solid copolymers. The method proposed to remove the homopolymer was based on the reported results for removing polymethylmethacrylate from a copolymer by washing with acetone (An, Yuan, Luo, & Wang, 2010). The copolymer (200 mg) was dispersed in acetone (50 mL) and placed under constant stirring for 48 h and the homopolymer was separated by filtration through a funnel with a fine-porosity sintered-glass plate. The extracted homopolymer was freeze-dried and quantified. The yield of purified copolymers was calculated in relation to the mass of unpurified copolymers. 2.3. Proton nuclear magnetic resonance (1 H NMR) Proton spectra of a 4% (w/v) solution of CPl purified in D2 O at 25 and 70 ◦ C were recorded on a Fourier transform Bruker Avance DRX 500 spectrometer. Sodium 2,2-dimethylsilapentane5-sulfonate (DSS) was used as the internal standard (0.00 ppm for 1 H NMR).

2.4. Critical association concentration (CAC) The CAC was determined by fluorescence spectroscopy using a methodology described in the literature (Patrizi, Piantanida, Coluzza, & Masci, 2009). The solutions of the copolymers in concentrations of 0.002–1.0 mg/mL were prepared in an aqueous solution of pyrene 5 × 10−7 mol/L. Fluorescence spectra were obtained using a QuantaMaster50 spectrometer (Photon Technology International) equipped with a thermostatic bath (Cole Parmer Polystat) linked to the cell compartment. The excitation spectrum (310–360 nm) was obtained using an emission wavelength (␭em ) of 374 nm. The intensity ratio (I338 /I334 ) was used to determine the CAC. A slit width of 0.5 mm (entrance and exit) was used for all measurements. The experiments were performed at 25 and 50 ◦ C.

C.M.W.S. Abreu et al. / Carbohydrate Polymers 154 (2016) 77–85

In the experiments performed at 50 ◦ C, the solutions were heated for 5 min prior to obtaining the spectra. 2.5. Fourier transform infrared (FTIR) spectroscopy The infrared spectroscopy analysis of the homopolymer and copolymers was conducted in KBr pellets on an FT-IR Shimadzu 8300 spectrophotometer in the region of 4000–400 cm−1 .

CG:NIPA ratio

1:0.5

1:1

1:2

Reaction Yield (%)a Purified Copolymer Yield (%)b Homopolymer Yield (%) CAC 25 ◦ C (mg/mL) CAC 50 ◦ C (mg/mL) LCST

75.8 ± 1.6 94 ± 1 6±1 0.47 0.17 37

94.3 ± 4.5 74 ± 2 26 ± 2 0.16 0.06 35

42.1 ± 2.3 65.5 ± 9 34.5 ± 9 0.63 0.31 36

b

Thermogravimetric analysis (TGA) measurements were carried out under synthetic air flow using TGA-Q 50 equipment (TA Instruments), in the temperature range 25–700 ◦ C, with heating rate of 10 ◦ C/min. 2.7. Epirubicin association efficiency and loading capacity The copolymer with a CG:NIPA ratio of 1:1 (64 mg) was stirred overnight in 32 mL of ultrapure water. Epirubicin hydrochloride (33.2 mg) was stirred in 3 mL of DMF and 5 equivalents of triehtylamine (40 ␮L) were added to remove the hydrochloride (based on Zhang et al., 2009). After stirring in the dark for 24 h, this solution was mixed with the copolymer solution at 37 ◦ C. The mixture was stirred in the dark at 40 ◦ C for 24 h, transferred to a dialysis bag and then dialyzed against ultrapure water for 48 h, after which the loaded nanoparticles were freeze-dried. The dried material was dissolved in DMSO and analyzed in a UV spectrophotometer (Shimadzu, UV-1800) at 483 nm. AE (Association Efficiency) =

LC (Loading Capacity) =

Table 1 Yield and characteristics of copolymers obtained with different CG:NIPA ratios.

a

2.6. Thermal analysis

total loaded drug weight x100% initial drug weight

loaded drug weight x100% nanoparticle weight

79

In relation to initial weight of CG + NIPA. In relation to unpurified copolymer.

prepared by stirring the copolymers in supplemented medium for 24 h in concentrations of 0.01, 0.1 and 1 mg/mL. The plates were incubated at 37 ◦ C in a 5% CO2 humidified incubator for 24 h. The dispersions were then removed from the plates and 200 ␮L of MTT reagent solution were added to each well followed by incubation. The MTT reagent was dissolved in PBS (5 mg/mL) and this solution was diluted by a factor of 10 with the supplemented medium. After 4 h the reagent solution was removed and 200 ␮L of dimethylsulfoxide (DMSO) were added to each well followed by gentle stirring for 20 min. The absorbance of the DMSO dispersion was then measured at a wavelength of 570 nm in a microplate reader. The cell viability of each sample was expressed as a percentage of the absorbance measured for the negative control. Eight replicate wells were tested for each control and sample concentration, and six of them were considered in the reporting of the results. The supplemented medium was used as the negative control and Triton-x 2% as the positive control. Data are given as mean ± standard deviation. 2.11. Statistical analysis Statistical significance was tested applying the Student’s t-test and p values of less than 0.05 were considered significant.

2.8. Dynamic light scattering (DLS)

3. Results and discussion

In order to determine the LCST, the hydrodynamic diameter and the polydispersity index (PdI) distribution of the copolymer solutions (0.1% w/v) were determined by raising the temperature from 25 to 50 ◦ C with an equilibration interval of 3 min for each temperature. All size measurements of the scattered light were obtained using a Nano Zeta Sizer Malvern, Model ZS 3600 with a scattering angle of 173◦ and zeta potential at 17◦ . Each experiment was carried out in triplicate.

3.1. Synthesis and characterization of CG-NIPA copolymer

2.9. Cell culture The human colon adenocarcinoma cell line Caco-2 (ATCC) was used for passages 48–51. The mucous-secreting Caucasian colon adenocarcinoma cell line (HT29-MTX, kindly provided by Dr. T. Lesuffleur, INSERM U178, Villejuif, France) was used for passages 50–52. Cells were cultured in Dulbecco’s modified Eagle’s medium (D-MEM, high glucose) supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) non-essential amino acids and 1% antibiotic solution (penicillin/streptomycin) in a humidified incubator under a 5% CO2 /95% air atmosphere at 37 ◦ C. 2.10. Cytotoxicity assay To evaluate the potential cytotoxic effect of the nanoparticles on intestinal cells, cells were seeded onto 96-well microplates (200 ␮L) at cell concentrations of 3.5 × 104 cells/mL of Caco-2 cells or 2.5 × 104 cells/mL of HT29-MTX cells. After 24 h of incubation, the cells were treated with 200 ␮L of the nanoparticle dispersions

Copolymers of cashew gum and PNIPA were successfully synthesized by radical polymerization. The yields of the CGNIPA purified copolymers and homopolymers with different molar ratios of cashew gum monosaccharide and N-isopropylacrylamide units (CG:NIPA molar ratio) are shown in Table 1. The purified copolymer yield, in relation to the unpurified copolymer, decreased with increasing values for the CG:NIPA ratio. An increase in the proportion of NIPA in the reaction favored the formation of the homopolymer. Fig. 1 shows the FTIR spectra for the cashew gum, graft copolymers and PNIPA homopolymer. The cashew gum spectrum shows a broad band at 3379 cm−1 due to O H stretching vibrations, a small band at 2937 cm−1 attributed to C H stretching vibrations, and strong bands at 1150, 1080 and 1030 cm−1 , which are due to stretching vibrations of C O C from glucosidic bonds and O H bending from alcohols. The absorption at 1647 cm−1 is due to the O H scissor vibrations of bonded water molecules and the shoulders at ∼1600 and 1414 cm−1 can be attributed to asymmetrical and symmetrical COO vibrations of the uronic acid present in the gum (Silva, de Paula, & Feitosa, 2007; Paula, Gomes, & de Paula, 2002; Silva et al., 2004). The homopolymer removed in the synthesis shows bands characteristics of poly-isopropylacrylamide with characteristic absorption bands at 1647 cm−1 (amide I band), corresponding to the stretching vibration of the double bond between the carbon and oxygen of the amide group (-CONH-) of NIPA, and at 1550 cm−1

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1:2

Absorbance

1:1

1:0.5

CG

homopolymer 4000

3500

3000

2500

2000

1500

1000 Fig. 2.

-1

1

H NMR spectra obtained for purified copolymers at 25 ◦ C in D2 0.

Wavenumber (cm ) Fig. 1. FTIR spectra for CG, CGNIPA copolymers and PNIPA homopolymer.

(amide II band), resulting mainly from N H bending of the amide group (Hirashima & Suzuki, 2007). In the homopolymer purification process acetone is used to dissolve the homopolymer. This strategy could lead to the dissolution of highly grafted cashew gum copolymers. However, the absence of bands at 1080 and 1037 cm−1 (characteristic of polysaccharide) in the homopolymer residue spectrum suggests that the copolymer fractions with a very high degree of grafting were not eliminated by the purification methodology. The spectra obtained for the copolymers present bands characteristics of CG (at 3387, 2937, 1037, 1080 and 1115 cm−1 ) as well as bands at 1638 cm−1 , attributed to C O stretching (amide) in NIPA, and at 2972 cm−1 , attributed to the C H stretching in isopropylacrylamide. The copolymer with a CG:NIPA ratio of 1:1 also shows bands at 1550 cm−1 attributed to N H bending (amine) in NIPA and at 1456 cm−1 attributed to C N bending (Silva et al., 2007; Rejinold, Chennazhi, Nair, Tamura & Jayakumar, 2011; Zhang et al., 2009), which indicates that this is the copolymer with the highest degree of grafting. According to the literature, cashew gum has anomeric protons in the range of 4.35–4.95 ppm due to the presence of ␣-d-glucose (4.95 ppm), ␣-l-rhamnose (4.81 ppm), ␤-d-galactose (1 → 3) (4.69 and 4.43 ppm) and ␤-d-glucuronic acid (4.51 ppm) (Moura Neto et al., 2011). The H-2 to H-5 signals are overlapped in the regions of 3.4–4.0 ppm and a signal at 1.3 ppm is due to the methyl protons of rhamnose (Moura Neto et al., 2011). The 1 H NMR spectra obtained at 25 ◦ C for the purified CGNIPA copolymer at different molar ratios are shown in Fig. 2. The signal related to the polysaccharide is not clearly shown. It is possible to identify the H-1 from glucose at 4.93 ppm and the proton from methyl groups of rhamnose at 1.3 ppm. The signals related to isopropylacrylamide appear at 1.1 ppm (a in the isopropylacrylamide structure) due to the two methyl groups, and two other signals at 2.0 and 1.6 ppm due to CH2 and CH groups of the main chain (b and c, respectively, in the isopropylacrylamide structure) (Wang et al., 2002). The difference in

Table 2 Thermal gravimetric data for CG and NIPA in synthetic air. Sample

Tonset (◦ C)

CG CGNIPA 1:0.5 CGNIPA 1:1 CGNIPA 1:2

224 232 226 231

Tmáx (◦ C) I

II

III

IV

45 52 40 49

235 295 296 295

284 323 348 318

433 347 – 357

the 1.1 ppm and 4.93 ppm peak areas, seen in Fig. 2, indicates that the copolymer with the highest amount of NIPA inserted in the CG chain is that produced with the CG:NIPA ratio of 1:1. This was also verified by the results obtained in the FTIR analysis. The thermal behavior of the copolymers can be observed in the H NMR spectra obtained at 70 ◦ C (Fig. 3). The anomeric protons of CG can now be clearly visualized. However, the signals attributed to the resonance of the PNIPA chains decrease considerably with an increase in the temperature from 25 to 70 ◦ C, which can be attributed to the dehydration of the PNIPA chains. This indicates that as the temperature increases the heat induces the formation of self-assembled nanogels with the hydrophobic chains of NIPA in the core of nanogel (Lv et al., 2011). Thermal gravimetric and DTG curves for CG and CGNIPA derivatives are shown in Fig. 4. The first mass losses are due to sample moistures. The onset of thermal degradation temperatures for the derivatives (Table 2) are higher than that for CG. It is worth to notice that thermal decomposition temperatures for all systems are higher than the temperature used for pharmaceutical product sterilization (121 ◦ C) indicating that the copolymers can be sterilized prior to their use as drug system carriers without undesirable degradation. The degradation of CG and CGNIPA 1:2 and 1:0.5 occurred in three steps (events II, III and IV in Table 2), while the CGNIPA 1:1 exhibits only two degradation events (II and III). The degradation temperature of events II and III for the copolymers are higher than the ones observed for CG.

C.M.W.S. Abreu et al. / Carbohydrate Polymers 154 (2016) 77–85

1:0.5

1:1

1:2 6 Fig. 3.

1

5

4 3 2 Chemical Shift (ppm)

0

H NMR spectra obtained for purified copolymers at 70 ◦ C in D2 0.

100

CG GCNIPA 1:0.5 CGNIPA 1:1 CGNIPA 1:2

80

Mass (%)

1

60 40 20 0 0

100 200 300 400 500 600 700 800

Temperature (°C) Fig. 4. Thermal gravimetric curves of CG and NIPA in synthetic air.

3.2. Characterization of self-assembled nanoparticles Purified CG:NIPA copolymers can form a nanostructure by selfassembling as they are dispersed in distilled water or PBS solution, depending on their concentration. In order to determine the critical aggregation concentration (CAC), the excitation spectra were obtained for pyrene in different copolymer solution concentrations. Pyrene is strongly hydrophobic and its solubility in water is very low (2–3 mmol/L). In the presence of micelles and other molecular systems, pyrene is preferentially solubilized in the hydrophobic regions within these aggregates (Kalyanasundaram & Thomas, 1977) and a decrease is observed in the 334 nm band intensity of pyrene on increasing the copolymer concentration. This decrease

81

confirms the formation of micelles and internalization of pyrene (Patrizi, Diociaiuti, Capitani, & Masci, 2009; Patrizi, Piantanida et al., 2009). Fig. 5 shows the variation in the excitation spectra obtained for pyrene with different copolymer concentrations. The CAC was calculated using a plot of the intensity ratio (I338 /I334 ) as a function of the copolymer concentration at 25 and 50 ◦ C (Fig. 6). The CAC was determined as the concentration at which the I338 /I334 ratio starts to increase, as shown in Fig. 6 (Table 1 shows the CAC values at different temperatures). The graphs show the crossover point at both temperatures, indicating that the nanostructure was formed even at the lower temperature (25 ◦ C). This behavior has not been reported for nanostructures formed with other polysaccharides inserted in NIPA. At 25 ◦ C the CGNIPA copolymers show association with the formation of a nanostructure, because the PNIPA chains in the copolymers tend to aggregate through self-assembly (Lv et al., 2011; Yusa, Shimada, Mitsukami & Yamamoto, 2004; Verbrugghe et al., 2003). NIPA copolymers are usually reported to shows CCA above the LCST, however some authors found that when the PNIPA chain are short the copolymers shows aggragation at temperature lower then LCST (Luo, Yu, & Xu, 2012; Neradovic, Soga, Van Nostrum & Hennink, 2004; Tiktopulo et al., 1995; Xu, Luo, Shi, Liu, 2006). As far as we concern this behavior is reported for the frist time for copolymers of NIPA and polysaccharide. At 50 ◦ C, above the LCST, the dehydration of the PNIPA chains leads to micellar association at lower concentrations compared with the respective values at 25 ◦ C (Table 1). These results indicate the potential stability of the nanoparticles after in vivo administration, since the CAC decreased when the temperature increased. The lowest CAC value was obtained for the copolymer with a CG:NIPA ratio of 1:1, which is the sample with the highest amount of grafted PNIPA according to the FTIR and NMR results. This behavior was also observed for PNIPA-grafted dextran, which showed a decrease in the CAC from 0.096 to 0.031 mg/mL as the NIPA degree of substitution increased (Patrizi, Diociaiuti et al., 2009; Patrizi, Piantanida et al., 2009). The variation in the CAC values is related to the amount of PNIPA grafted onto each copolymer. The greater the amount of PNIPA grafted or the longer the side chain formed the more hydrophobic the copolymer becomes and the particle aggregation will occur at lower concentrations, resulting in lower CAC values. The CAC value for carboxymethylated galactomannan grafted with PNIPA (1.038 mg/mL) was higher than that obtained for CGNIPA copolymers, probably due to the stronger hydrophilic character provided by the carboxylic groups (Gupta, Ghute, & Badiger, 2011). The DLS analysis results were used to investigate the thermoinduced self-assembly properties of the nanoparticles in solution. The point at which an abrupt increase in the size occurs with an increase in temperature is known as the copolymer LCST, and it is considered to lie at the intersection of the straight line passing through the inflection point of the curve and the horizontal straight line passing through the points before the transition (Lv et al., 2011; Patrizi, Piantanida et al., 2009). Fig. 7 shows the change in the particle size and the polydispersity index (PdI) for the CGNIPA copolymers in different CG:NIPA molar ratios as the temperature increases and Table 1 shows the LCST values. This sharp increase in the particle size indicates that the small nanostructures associate, forming larger aggregates at this temperature. The LCST values for the three copolymers were similar (35 - 37 ◦ C). All LCST values are higher than that observed for the PNIPA homopolymer (around 32 ◦ C). This phenomenon is common for PNIPA copolymers with polysaccharides and it is due to the presence of the more hydrophilic monosaccharide segments, which shifts the phase transition temperature to higher values, since the polymer solubility is increased (Chaw et al., 2004; Lai & Hsieh, 2012; Patrizi, Piantanida et al., 2009). It is interesting to note a sharper

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Fig. 5. Variation in pyrene excitation spectra with different CGNIPA concentrations.

increase in the size of the nanoparticles of CG:NIPA in a 1:1 ratio. This may be explained by the higher amount of PNIPA grafted onto this copolymer, as verified by the FTIR spectroscopy results. The higher the amount of grafted PNIPA the greater the hydrophobicity of the copolymer will be. The size of the nanoparticles of the three copolymers in the temperature range below the LCST is similar for the copolymers with CG:NIPA molar ratios of 1:1 and 1:2 (21 ± 2 nm), but it decreases when the CG:NIPA ratio increases (CG:NIPA 1:0.5) (12± 1 nm). At 50 ◦ C (i.e., a temperature higher than the LCST), the 1:1 CG:NIPA is the copolymer with the smallest particle size (113 nm). As this is the copolymer with the highest amount of grafted PNIPA, the lower size could be due to dehydration of the NIPA blocks leading to a more compact structure (Yusa, Shimada, Mitsukami, Yamamoto, 2004). The PdI values for the nanoparticles below the LCST ranged from 0.3 to 0.6 which, although low, had a large standard deviation. As the temperature increased above the LCST, lower PdI and standard deviation values were obtained, due to the formation of a more organized system (Fig. 7c).

0.500

3.3. Epirubicin association efficiency and loading capacity Epirubicin hydrochloride (EPI-HCl) was used as a cancer drug model. In this form the drug is water soluble and in order to produce a high-load system the EPI-HCl was treated with TEA to produce the non-ionized EPI as proposed by (Zhang et al., 2009). The AE and LC values for the EPI in the CG-NIPA (1:1) nanostructures were 63.67 ± 1.4% and 21.75 ± 0.48%, respectively. Zhang et al. (2009) studied pullulan acetate nanoparticles and reported an increase in the AE values from 38.9% to 61.8% for the EPI drug with an increase in the pullulan acetate degree of substitution (DS) value from 2.71 to 3.0. The AE value for EPI-loaded CG-NIPA (1:1) is close to that obtained with a higher pullulan acetate DS. However, the drug loading result obtained in the study reported herein is 3.7 times higher than that observed by Zhang et al. (2009) with a high pullulan acetate DS. Ahn et al. (2014) used an alginate graft with NIPA for the incorporation of doxorubicin, an anthracycline similar to epirubicin used for cancer treatment. The authors reported that for a drug/copolymer ratio close to that used in this work the AE

0.765

0.9

1:0.5 50 °C 1:0.5 25 °C

1:2 50 °C 1:2 25 °C

1:1 50 °C 1:1 25 °C

0.750

0.8 0.475

I338/I334

I338/I334

0.450

0.6

CAC CAC

I338/I334

0.735

0.7

0.720 0.705

0.5

0.425

0.690 0.4 0.675

0.400 0.01

0.1

1

Concentration (mg/mL)

0.01

0.1

1

Concentration (mg/mL)

0.01

0.1

1

Concentration (mg/mL)

Fig. 6. I338 /I334 ratio for pyrene as a function of the copolymer concentration at 25 ◦ C and 50 ◦ C.

C.M.W.S. Abreu et al. / Carbohydrate Polymers 154 (2016) 77–85

83

15

160

a

10

5

1:0.5 1:1 1:2

0 25 27

32

mp er at

35 40

50

100

10

Te

45 1

ur e( °C )

29

120

Z-Ave (d.nm)

Mean volume (%)

20

b

1000

Diameter (nm)

80 1,0

c 1:0.5 1:1 1:2

0,8

40 PdI

0,6 0,4

0 25

30

35

40

45

50

0,2

Temperature (°C)

0,0 25

30

35

40

45

50

Temperature (°C) Fig. 7. Diameter of nanoparticles (a), hydrodynamic size distribution (b) and PdI (c) in relation to an increase in the temperature.

140

Caco-2 cell viability (%)

120

* *

*

*

**

100

0.01mg/mL 0.1mg/mL 1mg/mL

**

**

80 60 40 20

et al., 2013). The MTT test was performed with CG at the highest concentration investigated for the copolymers (1 mg/mL) and no significant toxicity toward the Caco-2 and HT29-MTX cells was observed (Fig. 8). These results show that for the CG the CC50 value is >1000 ␮g/mL. The cell viability values for the nanoparticles with different CG:NIPA ratios and in three different concentrations are similar to the negative control (i.e., cells grown in their respective media). This indicates that the CG-NIPA copolymers did not show any toxi-

* *

160

HT29-MTX Cell Viability (%)

and LC were, respectively, 60% and 24%, which is similar to the results obtained in this study. These preliminary results indicate that CG-NIPA copolymers have the potential for use in biomedical formulations as an epirubicin delivery matrix. The results for the cytotoxicity of the CG:NIPA copolymers and CG are shown in Fig. 8. All of the in vitro cell viability experiments were performed using two different intestinal cell lines (Caco-2 and HT29-MTX). The unmodified polysaccharide of cashew gum has been reported to be a non-cytotoxic material (Quelemes

140

**

* *

*

0.01mg/mL 0.1mg/mL 1mg/mL

**

* *

120 100 80 60 40 20 0

0

1:0.5

1:1

1:2

GC CG

1:0.5

1:1

1:2

GC CG

Fig. 8. Cell viability in tests on Caco-2 and HT29-MTX cells in the presence of the copolymers at different concentrations. Error bars represent mean ± SD (n = 6); *p < 0.05 and **p < 0.01, when compared to cells incubated with only the medium.

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city toward the two cell lines even at the highest test concentration. These results are important considering the potential for the application of these copolymers in oral delivery systems. For the CG-NIPA copolymers and the CG the cell viability was higher in the case of the HT29-MTX cells compared to the Caco-2 cells at the same concentration. This could be because the mucus layer produced by HT29-MTX cells provides protection (Araújo & Sarmento, 2013). 4. Conclusions Graft copolymers of cashew gum/isopropylacrylamide were successfully synthesized and characterized. The copolymers formed nanoparticles at room temperature through self-assembly as they were dispersed in distilled water and they showed thermal responsiveness, with higher LCST values (closer to body temperature) being observed in comparison with PNIPA. The cell viability in the presence of the nanoparticles was good and the CG:NIPA graft copolymer shows good potential for application as a epirubicin delivery matrix. Acknowledgments The authors are grateful to the Bioinorganic Laboratory and CENAURENM of the Federal University of Ceará (Brazil) for the fluorescence and NMR spectroscopy analysis. The authors also acknowledge the Brazilian governmental agencies CNPq, CAPES and FUNCAP for financial support. This research was also partially supported by CESPU/IINFACTS under the project NanoGum-CESPU2014. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carbpol.2016.08. 031. References An, J., Yuan, X., Luo, Q., & Wang, D. (2010). Preparation of chitosan-graft-(methyl methacrylate)/Ag nanocomposite with antimicrobial activity. Polymer International, 59(1), 62–70. Araújo, F., & Sarmento, B. (2013). Towards the characterization of an in vitro triple co-culture intestine cell model for permeability studies. International Journal of Pharmaceutics, 458(1), 128–134. Basu, A., Kunduru, K. R., Abtew, E., & Domb, A. J. (2015). Polysaccharide based conjugates for biomedical applications. Bioconjugate Chemistry, 26(7), 1396–1412. Bigot, J., Charleux, B., Cooke, G., Delattre, F., Fournier, D., Lyskawa, J., et al. (2010). Tetrathiafulvalene end-functionalized poly(N -isopropylacrylamide): A new class of amphiphilic polymer for the creation of multistimuli responsive micelles. Journal of the American Chemical Society, 132(31), 10796–10801. Chaw, C. S., Chooi, K. W., Liu, X. M., Tan, C. W., Wang, L., & Yang, Y. Y. (2004). Thermally responsive core-shell nanoparticles self-assembled from cholesteryl end-capped and grafted polyacrylamides: Drug incorporation and in vitro release. Biomaterials, 25(18), 4297–4308. Chen, Y., Chen, Y., Nan, J., Wang, C., & Chu, F. (2012). Hollow poly(N-isopropylacrylamide)-co-poly(acrylic acid) microgels with high loading capacity for drugs. Journal of Applied Polymer Science, 124, 4678–4685. Cunha, P. L. R., de Paula, R. C. M., & Feitosa, J. P. A. (2009). Polissacarídeos da Biodiversidade Brasileira: Uma Oportunidade De Transformar Conhecimento Em Valor Econômico. Química Nova, 32(3), 649–660. Das, B., Nayak, A. K., & Nanda, U. (2013). Topical gels of lidocaine HCl using cashew gum and Carbopol 940: Preparation and in vitro skin permeation. International Journal of Biological Macromolecules, 62, 514–517. De Paula, R. C. M., Heatley, F., & Budd, P. M. (1998). Characterization of Anacardium occidentale exudate polysaccharide. Polymer International, 45, 27–35. Fundueanu, G., Constantin, M., & Ascenzi, P. (2008). Preparation and characterization of pH- and temperature-sensitive pullulan microspheres for controlled release of drugs. Biomaterials, 29(18), 2767–2775. Fundueanu, G., Constantin, M., & Ascenzi, P. (2010). Poly(vinyl alcohol) microspheres with pH- and thermosensitive properties as temperature-controlled drug delivery. Acta Biomaterialia, 6(10), 3899–3907.

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