curcumin inclusion complex

Journal Pre-proof Preparation, characterization and antitumor activity of a cationic starch-derivative membrane embedded with a β-cyclodextrin/ curcumin inclusion complex

Matheus S. Gularte, Rafael F.N. Quadrado, Nathalia S. Pedra, Mayara S.P. Soares, Natália P. Bona, Roselia M. Spanevello, André R. Fajardo PII:

S0141-8130(19)38470-3

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.01.104

Reference:

BIOMAC 14408

To appear in:

International Journal of Biological Macromolecules

Received date:

18 October 2019

Revised date:

22 November 2019

Accepted date:

9 January 2020

Please cite this article as: M.S. Gularte, R.F.N. Quadrado, N.S. Pedra, et al., Preparation, characterization and antitumor activity of a cationic starch-derivative membrane embedded with a β-cyclodextrin/curcumin inclusion complex, International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/j.ijbiomac.2020.01.104

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© 2020 Published by Elsevier.

Journal Pre-proof Preparation, characterization and antitumor activity of a cationic starchderivative membrane embedded with a β-cyclodextrin/curcumin inclusion complex Matheus S. Gulartea, Rafael F. N. Quadradoa, Nathalia S. Pedrab, Mayara S. P. Soaresb, Natália P. Bonab, Roselia M. Spanevellob, and André R. Fajardoa* a

Laboratório de Tecnologia e Desenvolvimento de Compósitos e Materiais Poliméricos (LaCoPol),

Universidade Federal de Pelotas (UFPel), Campus Capão do Leão s/n, 96010-900, Pelotas-RS, Brazil. b

Laboratório de Neuroquímica, Inflamação e Câncer (Neurocan), Universidade Federal de Pelotas

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(UFPel), Campus Capão do Leão s/n, 96010-900, Pelotas-RS, Brazil.

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Abstract

A membrane of cationic starch-derivative/poly(vinyl alcohol) was prepared and utilized

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as a support to immobilize a β-cyclodextrin/curcumin inclusion complex. The resulting

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material (denote as β-CD/CUR-MBN) was characterized in detail by different techniques. In vitro experiments revealed that β-CD/CUR-MBN enables the controlling

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of the curcumin release process, which is guided by the relaxation of the polymer matrix. Moreover, cytotoxic assays were performed to investigate the effect of β-

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CD/CUR-MBN on two cancer cell lines (melanoma and glioblastoma). The results showed that the polymeric membrane exerts higher cytotoxicity against these cells than

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free curcumin. Also, β-CD/CUR-MBN exerted a prolonged cytotoxic effect (up to 96 h), even using a low concentration (50 μg mL-1), indicating that the curcumin in the polymeric membrane showed increased bioavailability under the tested condition. βCD/CUR-MBN was non-cytotoxic against normal cells suggesting a specific action of this material against target cancer cells. The results reported here allow ranks βCD/CUR-MBN as a promising biomaterial to act as a local drug delivery system to treat cancer.

Keywords: Curcumin; cationic starch; inclusion complex; antitumor activity; melanoma; glioblastoma.

(*) Corresponding author: Prof. A.R. Fajardo, e-mail: [email protected] Phone: +55 53 3274-7356 - Fax: +55 53 3275-7354.

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Journal Pre-proof 1. Introduction Cancer includes a wide range of different diseases that arise as a result of the unregulated growth of malignant cells, which have the potential to invade or spread to other body parts leading to the formation of solid tumors [1]. Among these diseases, melanoma is a malicious malignancy and an aggressive form of skin cancer with high prevalence and resurgence indices [1,2]. Another common and deathly type of cancer is glioblastoma, which can cause a series of malignant brain tumors due to a genetic defect

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on astrocyte cells [1,3]. These two types of cancer are usually treated with similar

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approaches, which are based on the surgical resection of the tumors, followed by

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radiotherapy and chemotherapy [3]. However, these therapies are still not able to

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provide an efficient elimination of the tumor cells allowing in particular cases the resurgence of the tumors. Also, traditional chemotherapeutic drugs often exhibited a

lP

non-specific action and, consequently, the healthy cells are damaged due to their

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cytotoxic action [4]. Thus, new researches for less toxic drugs, as well as the development of most effective cancer treatments, are mandatory.

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In this sense, studies performed with curcumin (CUR), a natural yellow-pigment extracted from the rhizomes of the Curcuma longa [5], have demonstrated that this biocompound shows attractive biological and pharmacological activities against the propagation and proliferation of melanoma and glioblastoma [3,5,6]. Although CUR shows some efficiency in the treatment of tumors generated by melanoma and glioblastoma cancers, its limited water solubility (0.4 μg mL-1) in physiological condition impairs its application as an anticancer drug. The hydrophobic nature of CUR leads to a decrease in its bioavailability in biological systems, which reduces its uptake by the malignant cells [5,6]. One alternative to overcome such limitation is the formation of host-guest like complexes (i.e., inclusion complexes), where CUR can be

2

Journal Pre-proof accommodated inside a host molecule. As demonstrated in the literature, this strategy enhances the solubility, bioavailability, and stability of the guest molecule in the physiological environment [7]. Considering the different molecules that can be used as a host for CUR, cyclodextrins (CDs) offer various advantages as compared to other materials [8]. CDs are cyclic oligosaccharides in the shape of truncated cones made up of glucose units linked by 1→4 glycoside bonds [7]. In the aqueous environment, CDs exhibited a hydrophilic core, where the hydroxyl groups are located, and a hydrophobic

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cavity in which a variety of guest molecules can be hosted [8]. Among the CDs, the β-

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cyclodextrin (β-CD) is the most utilized for the preparation of inclusion complex due to

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its cavity that fits guest molecules with molecular weights between 200 and 800 g mol-1,

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which is the case of CUR [9].

Despite the beneficial aspects associated with the use of β-CD to form inclusion

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complexes with CUR, the efficiency of this kind of complex as a delivery system is still

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limited. Overall, the release of CUR from β-CD/CUR complexes is not restricted to a specific action site, which may cause systemic loss of the biocompound or undesired

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side-effects [7]. Alternatively, β-CD/CUR complexes can be embedded into polymeric supports that can be directly applied in specific sites (e.g., tumor site or surgical wound), providing a localized and sustained release of CUR [5]. According to some authors, this approach, known as a local drug delivery system (LDDS), can also prolong the therapeutic action of CUR on the treated site [4]. Such characteristics are paramount in cancer therapy because the LDDS can be placed, for example, in the surgical resection of the tumor and, then, provides a continuous release of the drug inhibiting the local tumor recurrence [10]. Under the past few years, the use of polymeric membranes and films as LDDS have been gained attention due to their versatility, flexibility, and an adequate form that provides a high coverage area for surgically implanted formulations

3

Journal Pre-proof or wounds caused by surgical tumor resection [10,11]. Generally, LDDS prepared with biopolymers usually show advantageous properties such as low-cost, non-toxicity, biocompatibility, biodegradability, and easy processing and mouldability [12,13]. Among the biopolymers applicable in the preparation of drug delivery systems, starch shows tremendous potential due to its physicochemical and biological properties [14,15]. Starch is a complex polysaccharide composed of two different macromolecules, amylose and amylopectin. In terms of processability, native starch shows some

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undesirable characteristics such as insolubility in cold water, excessive viscosity after

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heating, and tendency to retrogradation that limits its use [16]. The synthesis of starch-

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derivatives has been ranked as a promising strategy to overcome these limitations.

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Cationic starch (CSt) is a starch-derivative with positively charged groups in its backbone, which is commonly synthesized by the reaction of native starch with reagents

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containing amine, imine, sulphonium, phosphonium, or quaternary ammonium groups

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[17,18]. Beyond its high solubility in water and excellent membrane-forming properties, some studies have ascribed antibacterial and antifungal activities to CSt [19,20].

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Therefore, this study reports the preparation of an inclusion complex between CUR and β-CD (denoted as β-CD/CUR) and its subsequent immobilization in a polymeric membrane prepared by the blending of CSt and poly(vinyl alcohol) (PVA). PVA is a hydrophilic semi-crystalline, water-soluble, and non-toxic synthetic polymer that enables the preparation of the polymeric membranes by the freeze-thaw method, a physical crosslinking process that is free of toxic crosslinker agents [21]. We hypothesize that this system can enhance the bioavailability of CUR and, consequently, its antitumor activity. All prepared system was characterized in detail, while in vitro assays were performed to investigate the CUR-release from the β-CD/CUR-loaded

4

Journal Pre-proof membrane

(denoted

as

β-CD/CUR@MBN).

Also,

the

cytotoxicity

of

β-

CD/CUR@MBN against melanoma and glioblastoma cancer cell lines was investigated.

2. Materials and methods 2.1 Materials Rice starch (6% of amylose determined by Colossi et al. [16]) was kindly donated by LabGrãos/UFPel (Pelotas, Brazil). Poly(vinyl alcohol) (PVA, 98% and

Mw

of

124,000

g

mol-1),

of

hydrolyzed

(3-chloro-2-

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hydroxylpropyl)trimethylammonium chloride (CHPTAC), sodium hydroxide (NaOH),

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β-cyclodextrin (β-CD), and curcumin (CUR) from Curcuma longa (Turmeric) were

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purchased from Sigma-Aldrich (USA). Ethanol (P.A.) and hydrochloric acid (HCl) were

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without further purification.

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purchased from Synth (Brazil). All chemicals of analytical grade were used as received

2.2 Synthesis of the cationic starch-derivative (CSt)

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CSt was synthesized according to a previously reported protocol with a few modifications [21]. The general reaction route for the synthesis of CSt is shown in Scheme 1. Briefly, raw starch (1.5 g) was dissolved in deionized water (50 mL) in a reactor and, then, NaOH (10 g) was added. The system was stirred up to the mixture became homogenous. Next, CHPTAC (1.85 g) was added, and the pH of the solution was adjusted to pH 12 using a NaOH solution (1 mol L-1). The reaction was kept under stirring (300 rpm) at 60 °C in an oil bath. After 24 h, the reaction was cooled down to room temperature, and the CSt was precipitated using ethanol (100 mL). Later, the CSt was recuperated by vacuum filtration, neutralized with HCl (1 mol L-1), and washed with ethanol twice to remove the unreacted chemicals. The purified CSt was allowed to

5

Journal Pre-proof dry in an oven at 50 °C (overnight). The nitrogen content (N) of CSt was determined by the Kjeldahl method [20], and the degree of substitution (DS) was calculated per Equation (1):

𝐷𝑆 =

162 𝑥 𝑁 1400 − (154.64 𝑥 𝑁)

(1)

of

where 162 is the molecular weight of the anhydroglucoside unit of starch (g mol-1), N

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(%) is the nitrogen content of the CSt sample determined by the Kjeldahl method, and

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154.64 is the molecular weight of the CHPTAC (g mol-1). The DS determination was performed in triplicate. Thus, using Equation (1), the average value of DS for the as-

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synthesized CSt was calculated to be 0.7.

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Scheme 1

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2.3 Preparation of the β-CD/CUR inclusion complex The β-CD/CUR inclusion complex was prepared according to the method proposed by Gerola et al. [7]. A CUR stock solution was prepared in acetone (5 mg mL-1); thus, an aliquot of this solution was added to an aqueous solution of β-CD (25 mL, pH 5) with different CUR:β-CD molar ratios (1:2, 1:4, and 1:6). The resultant mixture was placed in an ultrasonic bath for 5 min to ensure the homogeneity of the system. Next, the mixture was maintained under vigorous and unceasingly stirring (200 rpm) at room temperature for 48 h. The final concentration of CUR in the aqueous medium was 40 μg mL-1. The complexation of CUR into the β-CD cavity was monitored by a Perkin Elmer Lambda 25 UV-vis spectrometer (USA) in the wavelength range of 200–400 nm. 6

Journal Pre-proof For this, an aliquot of the supernatant was collected in specific time intervals, and its UV-vis absorption was measured. Then, using an analytical curve (R2 ≈ 0.995), the concentration of CUR remaining in the supernatant was determined. The following equation calculated the loading efficiency (LE):

[𝐶𝑢𝑟]0 − [𝐶𝑢𝑟]𝑡 𝑥 100 [𝐶𝑢𝑟]0

(2)

of

𝐿𝐸 (%) =

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where [𝐶𝑢𝑟]0 the initial is the amount of CUR in solution and [𝐶𝑢𝑟]𝑡 is the amount of

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CUR remaining in the supernatant.

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2.4 Preparation of the β-CD/CUR@MBN membrane

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The polymeric membrane embedded with the β-CD/CUR was prepared according to the following procedure. Firstly, CSt (100 mg) was solubilized in distilled

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water (5 mL) under magnetic stirring (250 rpm) at room temperature. Separately, PVA

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(1000 mg) was also solubilized in distilled water (15 mL) at 80 °C for 3 h. Later, CSt and PVA solutions were blended and homogenized under magnetic stirring for 30 min at room temperature. The pH of the resulting solutions was adjusted to 5 and, then, it was added to the suspension containing the β-CD/CUR (20 mL). Again, the pH was adjusted to 5. The system was gently stirred (100 rpm) for 30 min to obtain a homogeneous yellowish solution, which was poured in a Petri dish (round-plate shape 85 x 10 mm) and immediately frozen at -20 °C for 12 h. Next, the system was thawed at room temperature for 30 min. Five consecutive freeze-thaw cycles were performed (1 h of freezing and 30 min of thawing) up to obtain the membrane-like material [22]. Finally, the β-CD/CUR-loaded membrane (denoted as β-CD/CUR@MBN) was recovered, purified distilled water, and dried under vacuum. It is worth to mention that 7

Journal Pre-proof the distilled water used to purify the membrane was further analyzed by UV-vis spectroscopy. No absorption was noticed at 425 nm (wavelength used to detect CUR). For comparative purposes, a membrane without the β-CD/CUR complex was prepared using a similar protocol. This sample was denoted as MBN.

2.5 Characterization techniques Fourier transform infrared (FTIR) spectra were recorded in the range of 400-

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4000 cm-1 with 64 scans per sample using a Shimadzu Affinity spectrometer (Japan).

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Before spectra acquisition, the grounded samples were blended with KBr powder and

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pressed into discs. Thermogravimetric analyses (TGA) were performed using a

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Shimadzu DTG-60 Analyzer (Japan) under N2 atmosphere at a heating rate of 10 ºC min-1 and a temperature range of 25–600 ºC. X-ray diffraction (XRD) patterns were

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recorded using a Siemens D500 diffractometer (Germany) with Cu-Kα radiation (λ =

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0.154 Å) at 30 kV/20 mA and scanning rate of 0.8º min-1. The diffraction angle (2θ) was ranged from 5 to 70º. Scanning electron microscopy (SEM) images were obtained using

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a JEOL JSM-6610LC microscope (USA) operating at 15 kV. Before SEM visualization, the freeze-dried samples were gold-coated by sputtering.

2.6 Swelling experiments

Swelling experiments were performed to investigate the liquid uptake behavior of the MBN and β-CD/CUR-MBN samples. Briefly, dry samples were weighted and then placed into vials filled with 50 mL of PBS solution (pH 7.4). The set (samples + vials) was kept on an orbital shaker (100 rpm) at 37 °C. At pre-determined time intervals, the swollen membranes were withdrawn, blotted carefully with tissue paper to

8

Journal Pre-proof remove the surface adsorbed liquid, and, then, they were reweighted. The following equation calculated the swelling degree at different times:

𝑆𝑤𝑒𝑙𝑙𝑖𝑛𝑔 𝑑𝑒𝑔𝑟𝑒𝑒 (%) =

(𝑊𝑠 −𝑊𝑑 ) 𝑊𝑑

𝑥 100

(3)

where Wd is the initial dry weight of the membranes, and Ws is the weight of the swollen

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membranes in different time intervals. These experiments were realized in triplicate.

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2.7 In vitro CUR-release experiments

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The CUR release profile was investigated using an ethanol-PBS solution as

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release media. Here, the ethanol:PBS volumetric ratio was fixed at 10:90 v/v-%, while

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the pH and temperature were set to 7.4 and 37 ºC, respectively [3]. For these experiments, β-CD/CUR@MBN samples (100 mg of the membrane loaded with 90 μg

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of CUR in its complexed form) were soaked into the release media, which has kept under mild stirring (100 rpm). At pre-determined time intervals, an aliquot (4 mL) of

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the release media was withdrawn, and its absorbance at 425 nm was measured using an UV-vis spectrometer. An equal volume (4 mL) of fresh release media was refilled in the system. The amount of CUR in the release media was determined using a calibration curve (R2 ≈ 0.998) that was built using standard concentrations of CUR in ethanol-PBS solution. All measurements were performed in triplicate.

2.8 In vitro biological experiments 2.8.1 Cell cultures Rat C6 glioblastoma and mouse B16F10 melanoma cell lines were obtained from American Type Cell Collection (USA). Cells were grown in culture flasks and 9

Journal Pre-proof maintained in 1% Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, UK) at pH 7.4 containing 8.4 mmol L-1 HEPES, 23.8 mmol L-1 NaHCO3, 0.1% fungizone, 0.5 U mL-1 penicillin/streptomycin and supplemented with 10% of Fetal Bovine Serum (FBS).

2.8.2 Primary astrocyte cultures Astrocyte cultures were prepared according to the protocol described by Gottfried et al. [23]. Briefly, cortex of newborn Wistar rats (1–3 days old) were

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removed and dissociated mechanically in Ca+2 and Mg+2-free balanced salt solution

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(CMF) (pH 7.64) containing 137 mmol L-1 NaCl, 5.36 mmol L-1 KCl, 0.27 mmol L-1

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Na2HPO4, 1.1 mmol L-1 KH2PO4, and 6.1 mmol L-1 of glucose. Dissociated tissue was

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subjected to centrifugation at 1,000 g for 5 min. After that, the pellet was suspended in DMEM (pH 7.4) supplemented with 10% of FBS. Then, cells (density ≈ 10×105) were

lP

seeded in poly-L-lysine-coated 48-well plates. Cultures were allowed to grow to

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confluence by 15–20 days, and the media was replaced every 5 days. All procedures used in the present study followed the “Principles of Laboratory Animal Care” of the

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National Institutes of Health (NIH) and were approved by the Ethical Committee of UFPel (CEEA 31292-2018).

2.8.3 In vitro cell culture treatment Glioblastoma cell line C6 and melanoma cell line B16F10 were seeded at 20×104 cells (48-well plates) for cytotoxicity experiments and allowed to grow for 24 h. Astrocyte cultures were prepared as described above. Cell cultures were treated with pure CUR, MBN, and β-CD/CUR@MBN (50, 100, and 200 µg mL-1 of encapsulated CUR considering its final concentration in the medium) for 24, 48, 72, and 96 h. The different amounts of CUR tested from the β-CD/CUR@MBN were adjusted, varying

10

Journal Pre-proof the membrane sample mass. The unloaded membrane samples (i.e., MBN) have the same mass of the loaded membrane samples. All the experiments were realized in triplicate.

2.8.4 Cell viability assay Dehydrogenases-dependent 3-(4,5-dimethyl)-2,5diphenyl tetrazolium bromide (MTT) reduction was used to estimate the viability of cell cultures according to the

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protocol described by Mosmman [24]. This method is based on the ability of viable

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cells to reduce MTT and form a blue formazan product. MTT solution was added to the

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incubation medium in the wells at a final concentration of 0.5 mg mL-1. The cells were

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left for 90 min at 37°C in a humidified 5% CO2 atmosphere. The medium was then removed, and the precipitate material was eluted with dimethyl sulfoxide (DMSO). The

lP

optical density of each well was measured at 492 nm using a SpectraMAX 190

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microplate reader (USA). Results were expressed as a percentage of the untreated cells (without free CUR or β-CD/CUR@MBN). The viability of the treated cells with the

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MBN sample is shown in the Supporting Information.

2.8.5 Statistical Analysis

Statistical analysis was performed using GraphPad Prism® 5 software. Data were expressed as mean ± standard error (S.E.M.) and were subjected to analysis of variance (ANOVA) followed by Tukey post-hoc test for multiple comparisons. Differences between mean values were considered significant when p < 0.05.

3. Results and discussion 3.1 Characterization of CSt

11

Journal Pre-proof The cationic starch (CSt) was synthesized through the etherification reaction between starch and CHPTAC in the presence of NaOH as a catalyst. Such reaction is based on the conversion of free hydroxyl groups in glucose units of starch to alkoxide groups due to the strong alkaline medium [20]. Then, alkoxide groups promote the nucleophilic attack of the CHPTAC molecule, and the hydroxyl groups of starch were etherified (see Scheme 1). The insertion of cationic groups into the starch backbone was investigated by

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FTIR analysis (Figure S1). The FTIR spectrum of the raw starch exhibited bands related

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to the O-H stretching of –OH groups (3380 cm-1), C-H stretching of –CH2 groups (2930

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cm-1), asymmetric vibration of C-O-C bond (1157 cm-1) and C-O vibration (1027 cm-1)

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of the glycosidic units (1027 cm-1) [18,25]. Also, a band ascribed to the absorbed water molecules on the starch was observed at 1646 cm-1. The spectrum obtained for CSt was

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similar to the spectrum of raw starch. However, the appearing of a new band at 1467

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cm-1 as noticed. This band is due to the C-N stretching of the (CH3)3N+ groups proceeding from the modifying agent [17]. Also, the O-H stretching band was shifted to

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a higher wavenumber region (centered at 3460 cm-1) due to the etherification of ‒OH groups and the dissociation of intra and intermolecular H-bonds of starch. The C-H stretching band was broadened likely due to the introduction of CHPTAC carbonic structure [25]. These finds corroborate the insertion of cationic moieties in starch due to the etherification of its free hydroxyl groups with the cationizing reagent. Finally, it is worth to mention that the FTIR spectrum of CSt did not show any band proceeding from free CHPTAC (usually observed at 1260 cm-1), suggesting that any toxic impurities (e.g., unreacted CHPTAC) are presented in the as-synthesized CSt. XRD measurements were performed to investigate changes in the crystallinity of starch after cationization reaction since chemical modifications usually promote

12

Journal Pre-proof changes in its crystalline structure. In the XRD pattern of raw starch, sharp crystalline peaks were observed at 2θ ≈ 15° and 23°, while an unresolved double peak was observed around to

2θ ≈ 17° and 18° (Figure S2). These diffraction peaks are

associated with the A-type crystalline structure of starch [26,27]. Overall, this crystallinity arises from crystallites formed by starch chains that interact by substantial intra and/or intermolecular H-bonds between their hydroxyl groups [27]. In contrast, the CSt diffraction pattern exhibited a broad peak, and none of the typical crystalline peaks

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of starch was noticed (Figure S2). Two small peaks at 2θ ≈ 31° and 45° are also

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observable in its XRD pattern, which correspond to trapped NaCl crystals between the

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CSt chains from the neutralization process [28]. In general, this data suggests that the

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crystalline structure of starch was changed after the cationization reaction. As aforementioned, the crystalline structure of starch results of intra- and intermolecular H-

lP

bonds between its hydroxyl groups. The etherification of these groups weakens these H-

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bonds affecting the starch crystallinity. Besides that, the strong alkaline condition used

starch [29].

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in the cationization reaction also promotes the disruption of the crystalline domains of

SEM images obtained from raw starch and CSt also corroborated this discussion. As observed from these images, the granules of raw starch showed a spherical shape with a smooth surface (Figure S3a), whereas the CSt showed the disruption and coalescence of the starch granules (Figure S3b), which confirms that the cationization reaction affects the arrangement of starch granules, as also their crystalline structure.

3.2 Characterization of the β-CD/CUR complex As related in the literature, the supramolecular structure of β-CD offers a hydrophobic cavity to host the CUR molecule forming a stable inclusion complex with

13

Journal Pre-proof enhanced solubility in water [8]. Here, inclusion complexes were prepared by adding CUR to β-CD solutions, varying the β-CD/CUR molar ratio (1:2, 1:4, and 1:6). Also, the same experiment was carried out for CUR in the absence of β-CD (as a control reaction). In all cases, the formation of the complex was monitored by UV-vis spectroscopy. According to the spectra depicted in Figure 1a-c, a continuous decrease in the intensity of the absorbance band of CUR (at 425 nm) was noticed for all reactions performed in the presence of β-CD.

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Conversely, for the control experiment, the absorbance band of CUR remained

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unaltered even after a few hours (Figure 1d). This result indicates that there is no

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noticeable degradation of CUR under the tested experimental conditions [30]. Under the

re

tested pH condition (pH 5), the CUR molecules remain in its β-keto-enolic configuration favoring the appearing of intra- and intermolecular H-bonds that drive the

lP

formation of stable CUR crystals [31]. When the β-CD is present in the medium, these

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CUR-CUR interactions are weakened due to the formation of the inclusion complex [32]. The inclusion complexation of CUR leads to the release of high-energy water

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molecules from the hydrophobic cavity of β-CD, which increases the entropy of the overall system and, as a consequence, such process is thermodynamically favorable [8,32]. Thereby, accordingly to these considerations, the decrease of CUR absorbance must be due to the inclusion complexation with the β-CD hydrophobic cavity.

Figure 1

The LE of CUR in the inclusion complexes was calculated for the different experimental conditions. It was found that LE increases according to the increase of CUR:β-CD molar ratio. In general lines, LE values increased from 65% to 93% when

14

Journal Pre-proof the CUR:β-CD ratio increased from 1:2 to 1:6, respectively. This result can be explained examining the CUR molecule dimension, which is too large (ca. 19 Å long and 6 Å wide) to be included entirely into a single β-CD cavity (ca. 7.8 Å wide). So, it is plausible to consider the complexation of CUR with two or more molecules of β-CD, whereas each phenolic ring of CUR occupies one hydrophobic cavity. Therefore, the need for a higher CUR:β-CD molar ratio for a complete complexation of CUR would be expected. According to the literature, other types of CUR reservoirs can be formed due

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to the non-inclusion complexation or micellar-like effects between CUR and β-CD,

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resulting in self-assembly and nano-assembly complexes [33].

-p

The kinetics of the inclusion complexation was investigated to determinate the

re

formation constant (Kf) using the typical double reciprocal plot, which is expressed by

1

lP

Equation (3) [7]:

= (𝐴

1

0 −𝐴1 )𝐾𝑓 [β−CD]

na

𝐴−𝐴0

2

+𝐴

1

0 −𝐴1

(3)

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where A and A0 are the absorbance of CUR in the presence and absence of β-CD, respectively. A1 is the expected absorbance for a complete complexation of the guest molecule. From the plot (A – A0)-1 versus [β – CD]-2, the constant Kf can be calculated (data not shown). In this study, the value calculated of Kf was about to 3.15 x 105 mol L2

(R2 ≈ 0.999), which is consistent with an inclusion complexation mechanism involving

more than one β-CD molecule [34]. Besides, it is worth to note that this model is based on the following assumptions: (i) the inclusion complexation is a dynamic process; (ii) the β-CD is in high excess with respect to CUR and, thereby, its free and analytical concentrations are similar; (iii) the variations in the concentration are proportional to the complex concentration; and (iv) at high β-CD concentration, essentially, all CUR 15

Journal Pre-proof molecules are complexed [34]. Then, to prepare the LDDS, the β-CD/CUR inclusion complex with higher loading efficiency (1:6 molar ratio) was selected since it possesses more encapsulated curcumin.

3.3 Characterization of β-CD/CUR@MBN membrane The immobilization of the β-CD/CUR complex in the polymeric membrane can be advantageous to obtain an efficient LDDS. Herein, CSt and PVA were chosen as

of

starting materials to prepare a polymer membrane. The use of PVA enables the

ro

preparation of a physically crosslinked polymeric matrix by the freezing-thaw (FT)

-p

method, which avoids the use of potentially toxic chemical crosslinkers. The formation

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of this matrix follows a unique crosslink mechanism, where the gelatinized PVA segments cease its motion with the water freezing [35]. As the size of water crystals

lP

increases, with the decreasing of temperature, regions of high PVA concentration are

na

formed and, consequently, the approximated PVA chains interact by H-bonding between their pendant hydroxyl groups favoring the formation of crystallites. Such

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interactions remained intact under thawing, and with the repeated FT cycles, a strong physical crosslinked network is obtained with the PVA crystallites acting as junction points between the polymeric chains [36]. Herein, the β-CD/CUR complex was embedded in the membrane-forming solution (CSt/PVA mixture) before the FT cycles. This approach favors the interactions among the functional groups of the polymers and the inclusion complex favoring the stability in the final material [37]. Moreover, the direct addition approach guarantees a higher dispersion of the β-CD/CUR complex, and a homogenous matrix can be obtained.

16

Journal Pre-proof To gain insights about the interactions between the β-CD/CUR and the polymeric matrix, FTIR analysis was performed (Figure 2). The FTIR spectrum of pure CUR showed its characteristic bands at 3510 cm-1 (O-H stretching of ‒OH group on benzene ring), 1622 cm-1 (C=C and C=O stretching of inter-ring chain), 1600 cm-1 (C=C stretching of benzene ring), 1508 cm-1 (C-O and C-C in-plane bending), 1430 cm-1 (C-H bending of olefins) and 1280 cm-1 (aromatic C-O stretching). The bands in the fingerprint region (800-1180 cm-1) are assigned to C-O, C-C, and C-H out of plane

of

bending modes presented in CUR structure [38]. The spectrum of β-CD showed bands

ro

at 3385 cm-1 (O-H stretching vibration), 2927 cm-1 (C-H stretching), 1646 cm-1

-p

(occluded water molecules in the hydrophobic cavity), 1154 cm-1 (C-O stretching) and

re

1024 cm-1 (C-O-C stretching from glucose units) [8].

The spectrum recorded for MBN (unloaded membrane) exhibited some bands

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associated with its constituents (CSt and PVA). For instance, the broadband centered at

na

3446 cm-1 (O-H stretching from hydroxyl groups), bands in the 2982-2800 cm-1 region (C-H stretching vibration from alkyl moieties) and bands at 1628 cm-1 (water

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molecules), 1568 cm-1 (C=O stretching from residual acetyl groups in PVA backbone), 1467 cm-1 (C-N stretching from (CH3)3N+ groups of CSt), 1341 cm-1 (C-OH bending) and 1240 cm-1 (O-H bending) [39]. Also, some important bands were observed at the 1200–1000 cm-1 region. These bands are related to the C-O stretching of H-bonded hydroxyl groups in the crystalline region (1143 cm-1) and the O-H stretching of unbounded hydroxyl groups in the amorphous zones (1098 cm-1) of the polymeric matrix. The ratio between such bands (Icrystalline/Iamorphous) is often used as an indicator of the crystallinity of PVA-based materials prepared by the FT method [40]. In this case, this value was ca. 0.89.

17

Journal Pre-proof Figure 2

The β-CD/CUR@MBN spectrum was quite similar to the MBN despite some discrepancies (Figure 2). As compared to the MBN spectrum, the band assigned to the O-H stretching was narrowed, while the bands related to the bending modes of O-H and C-OH bonds were shifted to a lower wavenumber region (1334 cm-1 and 1234 cm-1). These finds suggest the occurrence of H-bonds between the hydroxyl groups of the

of

polymer matrix and the inclusion complex. The band related to the cationic groups of

ro

CSt was also shifted to a lower wavenumber region (1455 cm-1) likely due to the

-p

electrostatic interaction between the β-CD/CUR complex and the negatively charged

re

moieties of the polymeric matrix [37]. Also, the ratio between the crystalline and amorphous bands of PVA decreased from 0.89 to 0.37 after the immobilization of β-

lP

CD/CUR complex, suggesting a reduction of the crystallinity of β-CD/CUR@MBN as

na

compared to MBN. Overall, the H-bonding between pendants hydroxyl groups of each PVA chain causes the crystallization of the PVA segments during the FT cycles. Yet,

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the immobilization of the β-CD/CUR complex impairs the PVA-PVA intermolecular interactions reducing its crystalline domains. These finds confirm that the β-CD/CUR complex was efficiently embedded in the CSt/PVA membrane. XRD patterns obtained for pure CUR, β-CD, MBN, and β-CD/CUR@MBN are shown in Figure 3. The diffraction pattern of pure CUR exhibited peaks at 2θ ≈ 12.2° (103/11-1), 14.4° (112/202), 21.1° (30-1), 23.8° (114), 24.4° (020), and 25.5° (120) confirming its crystalline nature [8,41,42]. The presence of other crystalline peaks in the CUR diffraction pattern, beyond those already mentioned, suggests that the CUR exists in its nanocrystalline form too [41]. The diffraction pattern of the β-CD also exhibited several sharp peaks and an intense peak centered at 2θ ≈ 12.6° (041) [43]. This type of

18

Journal Pre-proof diffraction pattern indicates a high crystallinity, and it is characteristic of a cage-type packing structure in which the cavity of each β-CD molecule is blocked by neighboring β-CD molecules [44]. The diffraction pattern of the MBN presented a sharp crystalline peak at 2θ ≈ 19.4° (101) and two less intense peaks centered at 2θ ≈ 22.8° (200) and 40.6° (102), respectively. These peaks are assigned to the PVA crystalline microdomains in the polymeric matrix, which are formed during the FT cycles, and they act as physical crosslinking points through the matrix [45]. It can be inferred from this

of

XRD data that the CSt does not affect the formation of the PVA crystalline

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microdomains negatively, although it could interact with the PVA chains as

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demonstrated by FTIR data [45].

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The β-CD/CUR@MBN diffraction pattern exhibited peaks at the same 2θ angles than MBN, whereas the intensity of the peaks was lower (see Figure S4). This result

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suggests that the crystallinity of the β-CD/CUR@MBN decreases due to the

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immobilization of the complex. In order to confirm this hypothesis, the crystallinity (Xc) of both membranes was estimated using the Equation (4). According to this equation,

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the ratio between the area of the crystalline peaks (A1) and the area subtending the full diffraction profile of the samples (A2) gives the crystallinity data [45].

𝑋𝑐 (%) =

𝐴1 𝑥 100 𝐴2

(4)

The values of Xc estimated for MBN, and the β-CD/CUR@MBN were 48% and 34%, respectively, which confirms our suggestion that the complex affects the crystallinity of the polymeric membrane and corroborates the FTIR data. As additional information, the diffraction pattern of the β-CD/CUR@MBN did not exhibit any characteristic diffraction peak of CUR and β-CD. According to the literature, the 19

Journal Pre-proof complexation of the CUR molecule into the hydrophobic cavity of the β-CD results in an amorphous material [8,44,46]. This behavior could be a result of the formation of a channel package structure involving one molecule of CUR and two or more β-CD molecules [46]. In the channel package structure, the β-CD molecules are stacked on top of each other to form long cylindrical channels in which the CUR molecule resides. Overall, the orientation of these channels is head-to-head, tail-to-head, or tail-to-tail that

to be in consonance with our experimental results.

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results in a three-molecule repeating unit at each β-CD stack [46]. Such statements seem

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The thermal properties of CUR, β-CD, MBN, and β-CD/CUR@MBN were

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investigated since they provide information about the chemical and structural nature of

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the prepared materials. TGA curves obtained for these samples are shown in Figure 4. As assessed, pure CUR exhibited the first weight-loss stage (⁓6% of weight loss) in a

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temperature range of 37‒139 °C due to the moisture evaporation and the

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dehydroxylation of its hydroxyl groups [7]. Next, the carbonic structure of CUR is thermally degraded, which explains the principal weight-loss stage (⁓65% of weight

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loss) with a maximum at 283 °C [47]. The TGA curve of β-CD showed two weight loss too. The first (⁓10% of weight loss) occurred between 40–105 °C due to the evaporation of bonded water molecules, while the second (⁓ 85% of weight loss) with a maximum at 120 °C is ascribed to the sample decomposition [8,48]. Comparing the TGA curves of the MBN and β-CD/CUR@MBN, both samples presented three main stages of weight loss. For the pristine membrane, the first stage (⁓4% of weight loss) occurred in the temperature range of 73‒195 °C due to evaporation of absorbed water, while the second stage (⁓66% of weight loss) with a maximum at 279 °C is related to the depolymerization of the polymeric chains of PVA and CS leading to the formation of simplest compounds (e.g., carbonaceous compounds). The thermal degradation of

20

Journal Pre-proof these compounds is related to the third stage of weight loss (⁓80%), which has a maximum at 435 °C [49]. For the β-CD/CUR@MBN sample, the weight loss of each stage is more pronounced than the pristine membrane, and its degradation temperatures were shifted to lower temperatures, as evidenced by the DTG analysis (Figure S5). As aforementioned, the β-CD/CUR complex exerts a noticeable effect on the structure of the polymeric matrix (e.g., decreasing the membrane crystallinity) because of its interaction (H-bonding and electrostatic interaction) with the functional groups of the

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PVA and CSt. Consequently, β-CD/CUR@MBN is more susceptible to thermal

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degradation than MBN [50]. This data aggress with the data collected from the FTIR

re

-p

and XRD analysis.

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Figure 4

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The morphology of the MBN and β-CD/CUR@MBN membranes was examined by SEM microscopy. MBN showed a homogenous and rough surface without visible

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phase separation, suggesting high compatibility between the CSt and PVA (Figure 5a). The dense morphology of this membrane and the absence of porous suggest that the crosslinking process and the drying process caused the packing of the polymer chains. Comparatively, the CUR-loaded membrane presented a wrinkled surface; however, again, it was not observed an apparent phase separation (Figure 5b). This morphological discrepancy between the two samples is highlighted at higher magnification (Figures 5c and 5d). Overall, the distinct surface morphology of β-CD/CUR@MBN can be attributed to the presence of the β-CD/CUR complex since it interacts with the polymeric chains affecting the crosslinking process. Such interactions can create zones

21

Journal Pre-proof with a higher concentration of polymeric chains surrounding the β-CD/CUR complex, which may explain the wrinkled surface of the β-CD/CUR@MBN membrane.

Figure 5

A desired feature of an efficient LDDS is its ability to absorb and retain the exudate fluid secreted from the wound, such as in skin cancer lesion [13]. An adequate

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hydrophilic/hydrophobic balance is also paramount for an LDDS since this kind of

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biomaterials can serve as an implantable device at the edge of the tumor or as an active

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bandage in the surgical resection treatment of solid tumors [4]. Also, the liquid uptake

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process is closely related to the release of active drugs loaded in the LDDS [51]. Here, swelling experiments were performed in order to investigate the liquid uptake capacity

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of the MBN and β-CD/CUR@MBN when exposed to PBS medium (pH 7.4, 37 °C).

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The swelling curves are depicted in Figure 6.

Figure 6

As observed, the swelling kinetics of both membranes exhibited a similar trend at the beginning of the experiment. However, after 20 min, a discrepant behavior between the two membranes was noticed. For MBN, the swelling process slowdown after 30 min, and the maximum swelling degree (197%) is achieved in 120 min. After this while, the swelling process tends to leave off. In contrast, the liquid uptake process seems to last more time for the β-CD/CUR@MBN sample. The maximum swelling (247%) is achieved only after 150 min after the beginning of the experiment. After that, the equilibrium is reached. Despite such discrepancies related to the swelling kinetics, it

22

Journal Pre-proof should be highlighted too that the β-CD/CUR@MBN samples absorbed 50% more liquid than the pristine membrane. For both samples, the liquid uptake ability can be assigned to the hydrophilic nature of the polymers, CSt and PVA. These two polymers have hydrophilic groups available to interact with the water molecules [52]. Despite the noticeable hydrophilicity of exhibited by both membranes in PBS (pH 7.4), the higher swelling ability of β-CD/CUR@MBN is caused by the complex immobilized in its polymer matrix. The low crystallinity of β-CD/CUR@MBN as compared to MBN

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facilitates the expansion of the polymeric matrix during the swelling process, which is

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benign to the liquid uptake. In a more expansible matrix, the water molecules are

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accommodated easily between the polymeric chains, which result in a higher swelling

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3.4 In vitro CUR-release

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this material as a promising LDDS.

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degree [45]. The remarkable swelling ability of the β-CD/CUR@MBN membrane ranks

In order to investigate the potential of the β-CD/CUR@MBN membrane as an

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efficient LDDS, the CUR-release profile was investigated in ethanol-PBS solution (pH 7.4) at 37 °C [3,4]. This release medium was used to induce the dissociation of CUR from the β-CD/CUR complex. As aforementioned, the hydrophobic cavity of the β-CD offers favorable ambient to the CUR molecules, while the hydrophilic core enhances the solubility of CUR in aqueous mediums. However, as a result of the inclusion complexation of CUR into the β-CD cavities, the characteristic UV-vis absorbance profile of CUR is altered [53]. So, the release of CUR from the complex directly in the PBS medium cannot be investigated by UV-vis spectrometry. Thus, a small portion of ethanol was added to the PBS solution since it could act as a solubilizing and stabilizing agent that can promote the dissociation of CUR from the inclusion complex enabling its

23

Journal Pre-proof detection by UV-vis spectroscopy [54]. This strategy has been widely utilized by several researchers [55-57].

Figure 7

The cumulative release of CUR from β-CD/CUR@MBN as a function of time is shown in Figure 7. Also, for comparative purposes, the release of free CUR (i.e., not

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encapsulated) was investigated. As observed, β-CD/CUR@MBN was able to promote a

ro

controlled release of CUR, preventing an initial burst releasing during the first hours of

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the experiment. In contrast, 100% of the free CUR was released before the 3rd hour

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after the beginning of the experiment. At this same time interval, only 6% of the encapsulated CUR was released from β-CD/CUR@MBN (see the inset in Figure 7).

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Burst release leads to a higher initial amount of drug delivered, which may cause some

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inconveniences for the patients, such as an unspecific action of the drug that leads to local or systemic toxicity [13]. Besides that, β-CD/CUR@MBN sustains the CUR

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release profile, where the amount of CUR released increases linearly up to achieve the equilibrium, which occurs around 36 h. At the end of the experiment, the cumulative release of CUR from the loaded membrane was 85%. From a practical viewpoint, this sustained CUR release profile is an attractive feature for a potential LDDS because it guarantees a high therapeutic and pharmaceutical efficiency of the CUR, which are vital characteristics to the treatment of aggressive cancers, such as melanoma [10]. The release of CUR from β-CD/CUR@MBN seems to be related to the hydrophilicity and the swelling of the polymeric matrix. This statement is coherent with our experimental data since the CUR release increases as the contact time of the membrane with the release medium increases. Hydrophilic groups of CSt and PVA interact with the water

24

Journal Pre-proof molecules promoting the swelling of the membrane, which favors the release of the βCD/CUR complex (consequently, the release of CUR) [58,59]. To gain further insights into the release mechanism of CUR from the βCD/CUR@MBN, the data depicted in Figure 7 were fitted using the Korsmeyer-Peppas release model (Equation (5)), which can be expressed in its linearized form (Equation

𝑄𝑡 ) = log 𝑘𝐾𝑃 + 𝑛 log 𝑡 𝑄∞

(5)

(6)

re

-p

log (

ro

𝑄𝑡 = 𝑘𝐾𝑃 𝑡 𝑛 𝑄∞

of

(6)):

lP

where 𝑄𝑡 and 𝑄∞ are the cumulative amounts of CUR released at time t and equilibrium, respectively, while 𝑘𝐾𝑃 and 𝑛 are fitting parameters. The parameter 𝑛

na

(dimensionless) is the release exponent of the Korsmeyer-Peppas model and gives relevant information about the release mechanism [7]. For the release of solutes from

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polymeric membranes, values of 𝑛 ≤ 0.5 indicate a diffusion-controlled release (Fickian or pseudo-Fickian) mechanism; 0.5 < 𝑛 < 1.0 suggest an anomalous mechanism and 𝑛 = 1.0 refers to a Non-Fickian (case II transport) release behavior. Higher values of 𝑛 (𝑛 > 1) occur when the drug release from polymeric membranes is based on a super case II transport mechanism [60]. It is worth to mention that the Korsmeyer-Peppas establishes an exponential correlation between the drug release and the time, and it predicts the first 60% of drug release [60]. Therefore, the parameters 𝑘𝐾𝑃 and 𝑛 were calculated from the regression plot of Equation (6) (Figure S6a), and their values are listed in Table 1.

Table 1 25

Journal Pre-proof

The data listed in Table 1 indicate that the experimental data were well fitted by the Korsmeyer-Peppas model (R2 = 0.992) and the analysis of the calculated value of 𝑛 (𝑛 = 0.960) reveals that the CUR release process followed an anomalous mechanism. According to the anomalous mechanism, the release of CUR from β-CD/CUR@MBN occurs due to the polymer macromolecular chain relaxation (i.e., swelling of the polymeric matrix) and, subsequently, Fickian transport [60]. Moreover, the erosion of

of

the polymeric matrix could also contribute to the CUR release [61]. This dependence of

ro

the CUR release with the swelling of the polymeric matrix can be elucidated by

-p

considering the interactions (electrostatic interactions and H-bonding) between the

re

inclusion complex and the matrix (such interactions were proved by the FTIR, XRD and TGA analysis). Since the inclusion complex interacts strongly with the polymer matrix,

lP

the release of CUR in the first stages of the experiment is low. However, as the

na

polymeric matrix swells, the distance between the polymeric chains increases weakening the interactions among the β-CD/CUR and the polymeric matrix. Therefore,

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as time goes on, more CUR is released from the membrane. The release kinetics of CUR from β-CD/CUR@MBN was also evaluated, fitting the experimental data with the first-order and second-order kinetic models, which are expressed by Equations (7) and (8) [62].

ln(𝑄𝑡 − 𝑄∞ ) = ln 𝑄∞ + 𝑘1 𝑡

(7)

𝑡 1 1 = + 𝑡 2 𝑄𝑡 𝑘2 (𝑄∞ ) 𝑄∞

(8)

where 𝑄𝑡 and 𝑄∞ have the same meaning as before, and 𝑘1 and 𝑘2 are the first-order (h1

) and second-order (mg h-1) kinetic constants, respectively. The regression plots were 26

Journal Pre-proof made for each equation (Figure S6b and S6c) and the calculated 𝑘1 and 𝑘2 are listed in Table 1. As noticed, the second-order model showed the highest correlation coefficient value (R2 = 0.979), which indicates that this model is reliable to explain the CUR release kinetics. According to this kinetic model, it can be supposed that the diffusion of the β-CD/CUR complex into the release medium is governed by the relaxation of the polymeric chains in the swollen matrix, thereby, facilitating the CUR release from β-

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3.5 In vitro cytotoxicity against cancer cell lines

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CD/CUR@MBN [7,63].

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Melanoma and glioblastoma are frequent malignancies, and both represent

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aggressive forms of cancer, where its advanced stage is characterized by a poor prognosis and a high incidence probability due to the lack of effective treatments [64].

lP

The conventional treatments for these types of cancer include surgery to surgical

na

resection of the solid tumor, which is followed by chemotherapy. Chemotherapeutic drugs are engineered to kill the tumor cells based on their toxicity; however, these drugs

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usually have a non-specific action inducing severe side effects [4]. CUR was proven to be an efficient biocompound in the treatment of melanoma as well as glioblastoma by acting in several pathways and target multiple genes, transcriptions factors, enzymes, and cell cycle proteins, leading to apoptosis of the tumor cells [64,65]. The toxic effect of CUR on unhealthy cells is based on membrane-mediated mechanisms, such as the activation of the enzymes caspases-3 and -8, inducing the death of the tumor cells [62]. Furthermore, compared to the traditional chemotherapeutic drugs, CUR possesses lower cytotoxicity against normal cells [63]. Despite these attractive properties, the use of free CUR in the treatment of melanoma and glioblastoma cancers is limited due to its small bioavailability under physiological conditions. Here, a β-CD/CUR inclusion complex

27

Journal Pre-proof was embedded in a polymeric matrix to potential LDDS application. This strategy allows, for example, controlling and sustaining the release of CUR in a specific treatment site, increasing its bioavailability and action in a physiological medium [4]. In vitro experiments were performed to assess the cytotoxic effect of the free CUR, MBN, and β-CD/CUR@MBN against melanoma and glioblastoma cell lines. The results concerning the cytotoxic effect against melanoma are displayed in Figure 8. Overall, both free CUR and β-CD/CUR@MBN exhibited a cytotoxic effect on the

of

melanoma cells, as compared to the MBN (the control, Figures S7a and S7b),

ro

confirming the anti-cancer activity ascribed to CUR. After 24 h of incubation, the

-p

viability of the melanoma cells treated with different concentrations of free CUR

re

decreased from 27.42% to 25.47% when the concentration increased from 50 μg mL-1 to 200 μg mL-1. In parallel, after 24 h, the viability of the melanoma cells decreased from

lP

66.22% to 58.93% when the concentration of β-CD/CUR@MBN increased from 50 to

na

200 μg mL-1 (Figure 8a). The increment of the incubation time reveals that the cytotoxic effect of free CUR on the melanoma cell line is neglected (see Figures 8b-d). On the

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other hand, longer incubation time increased the cytotoxicity of β-CD/CUR@MBN against the melanoma cells (Figures 8b-d) likely due to the sustained release of CUR (prolonged action). After 96 h of incubation, the viability of melanoma cells was below 12% for all tested concentrations of β-CD/CUR@MBN (Figure 8d).

Figure 8

In the case of the glioblastoma cell line, similar results were observed noticed (Figure9 a-d)). After 24 h, the viability of the glioblastoma cells incubated with free CUR varied from 26.06% (50 μg mL-1) to 34.14% (200 μg mL-1), while the viability of

28

Journal Pre-proof the cells incubated with β-CD/CUR@MBN decreased from 81.76% to 68.55% when the of the membrane concentration (Figure 9a). After 96 h of treatment, the viability of the glioblastoma cells was similar for all tested concentrations of free CUR (~87.00%) (Figure 9d). In contrast, after 96 h, the viability of glioblastoma cells incubated with βCD/CUR@MBN showed a remarkable decreasing, especially for the highest concentration of the membrane (200 μg mL-1). These in vitro experiments revealed that β-CD/CUR@MBN was more cytotoxic

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to both cancer cell lines than free CUR. Overall, glioblastoma cells exhibited a higher

ro

sensitivity to the action of the β-CD/CUR@MBN when compared to melanoma cells.

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This result is encouraging since it is known the difficulty of the traditional

re

chemotherapeutic drugs to combat the glioblastoma cells [3].

na

lP

Figure 9

It can be inferred from these results that free CUR has a fast cytotoxic effect on

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both cancer cell lines; however, it seems not to be able to prevent the proliferation of these cancerous cells effectively. On the other hand, the viability of the cancer cell lines treated with β-CD/CUR@MBN decreased continuously. When the free CUR is used, it can promptly exhibit its cytotoxic effect on the cells inducing their death. Despite that, as the free CUR presents a low solubility, its bioavailability is reduced, and its uptake by the cells is limited. As a result, the melanoma and glioblastoma cells are not damaged by the biological effect exerted by CUR under prolonged contact time [5]. Conversely, the protection of CUR against the biological degradation enabled by the inclusion complex is a relevant factor to assure its cytotoxic effect against cancer cells [66]. Besides that, the sustained release of CUR from β-CD/CUR@MBN guarantees a

29

Journal Pre-proof prolonged biological effect on the cancer cells, which decrease their viability, as shown by the experimental results depicted in Figures 8 and 9. The cytotoxicity effect of the free CUR, MBN (used as control), and βCD/CUR@MBN against normal cells (astrocyte cells) was investigated, and the results are shown in Figures 10a-d. As assessed, free CUR exerted some cytotoxicity against the tested normal cells, mainly for long incubation times and high CUR concentrations. Despite a now-linear behavior, the viability of normal cells decreased to the half

of

(~48.35%) after 96 h of incubation using a concentration of free CUR of 200 μg mL-1.

ro

These results suggest that the use of high concentrations of free CUR to treat cancer

-p

cells cause a non-selective cytotoxic effect on the astrocyte cells, thereby promoting

re

their death. On the other hand, β-CD/CUR@MBN exerted a neglected effect on the tested normal cells, as noticed in Figure 10a-d. For all tested conditions (incubation

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times and membrane concentrations), it was quantified that the cell viabilities were

na

higher than 100%, suggesting the polymeric membrane has a benign effect on the normal cell growth. This hypothesis is strengthened by the results obtained for the

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MBN, which was tested as a control (Figure S7c). The biocompatible property of CSt and PVA associated with other intrinsic features may favor cell growth. It is an attractive advantage since the β-CD/CUR@MBN could be used as LDDS to cover a wound favoring its healing too. The absence of cytotoxicity noticed to βCD/CUR@MBN can be ascribed to its sustained release ability that regulates the amount of CUR in contact with the normal cells (or healthy cells) [67,68].

Figure 10

30

Journal Pre-proof The results reported here indicate that β-CD/CUR@MBN shows an enhanced anti-cancer activity against melanoma and glioblastoma cells, and, thereby, it could be used as an efficient LDDS. It is worth to mention that different biomaterials have been developed to encapsulate and release CUR, such as microspheres and nanocarriers [5,6]. However, many of these CUR-containing biomaterials suffer from systemic distribution of the drug, reducing the CUR pharmacological action. Moreover, the cytotoxic effect of these materials on normal cells usually is not evaluated, or they present a non-

of

specific action [2,69]. On the contrary, the polymeric membrane prepared in this study

ro

seems to overcome these drawbacks. Also, the physical characteristics (i.e., high

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coverage area possibility and lower thickness) of the β-CD/CUR@MBN membrane

re

allow its use as an active dressing and/or implantable LDDS for post-surgical treatment

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4. Conclusion

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of surgical wounds caused by surgical tumor resection.

A polymeric membrane based on cationic starch and PVA was successfully

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prepared and tested as a vehicle for the controlled release of curcumin (CUR). Also, its antitumor activity was tested against two cancer cell lines (melanoma and glioblastoma). Firstly, CUR was complexed with β-cyclodextrin (β-CD) to form an inclusion complex to enhance its antitumor activity under physiological conditions. Next, the β-CD/CUR complex was embedded in the cationic starch/PVA membrane (βCD/CUR@MBN). As assessed, the presence of the complex in the polymeric matrix affects several of its properties. In vitro experiments showed that a CUR is released from β-CD/CUR@MBN in a controlled manner following second-order kinetics. Furthermore, the β-CD/CUR@MBN membrane reduced the cell availability of the two tested cell lines, while it was non-cytotoxic against normal cells. This result indicates a

31

Journal Pre-proof possible CUR-specific action guided by the polymeric membrane. Our findings suggest that this novel biomaterial can be a promising device to treat cancer locally.

Acknowledgments The authors are thankful to CNPq for the financial support (Grant. 404744/20184), scholarships, and for the PQ-fellowships to A.R.F. and R.M.S. This study was also financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -

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Brasil (CAPES) - Finance Code 001.

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Conflict of interest

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None.

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[4] M.Y. Guo, G.Q. Zhou, Z. Liu, J. Liu, J.L. Tang, Y.T. Xiao, W.S. Xu, Y. Liu, C.Y. Chen, Direct site-specific treatment of skin cancer using doxorubicin-loaded nanofibrous membranes, Sci. Bull. 63 (2018) 92-100. https://doi.org/10.1016/j.scib.2017.11.018.

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[5] Y.B. Sun, L.N. Du, Y.P. Liu, X. Li, M. Li, Y.G. Jin, X.H. Qian, Transdermal delivery of the in situ hydrogels of curcumin and its inclusion complexes of hydroxypropyl-beta-cyclodextrin for melanoma treatment, Int. J. Pharm. 469 (2014) 31-39. (2014) 31-39. https://doi.org/10.1016/j.ijpharm.2014.04.039.

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[6] A.G.B. Pereira, A.R. Fajardo, S. Nocchi, C.V. Nakamura, A.F. Rubira, E.C. Muniz, Starch-based microspheres for sustained-release of curcumin: Preparation and cytotoxic effect on tumor cells, Carbohyd. Polym. 98 (2013) 711-720. https://doi.org/10.1016/j.carbpol.2013.06.013.

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Figure captions Figure 1. UV-Vis spectra of CUR in the absence of the β-CD (a) in the beginning (black line) and at the end of the experiment (red line), and in the presence of β-CD with CUR:β-CD molar ratios of (b) 1:2, (c) 1:4, and (d) 1:6. 39

Journal Pre-proof Figure 2. FTIR spectrum of CUR, β-CD, CSt, PVA, MBN, and β-CD/CUR@MBN. Figure 3. XRD patterns of CUR, β-CD, MBN, and β-CD/CUR@MBN. Figure 4. TGA curves of CUR, β-CD, MBN, and β-CD/CUR@MBN. Figure 5. SEM images of the MBN (a and c) and β-CD/CUR@MBN (b and d) membranes. Figure 6. Swelling kinetics curves of the MBN and β-CD/CUR@MBN in PBS (pH 7.4) at 37°C.

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Figure 7. Cumulative release of CUR from the β-CD/CUR@MBN membrane in

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ethanol-PBS solution (pH 7.4) at 37 °C.

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Figure 8. Cell viability of the melanoma tumor cells treated with free CUR and the βCD/CUR@MBN at (a) 24 h, (b) 48 h, (c) 72 h, and (d) 96 h using different CUR

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concentrations (50, 100, and 200 μg mL-1). The results showed significant levels (p <

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0.05) when compared to the control (cells treated with the MBN). Figure 9. Cell viability of the glioblastoma tumor cells treated with free CUR and the βCD/CUR@MBN at (a) 24 h, (b) 48 h, (c) 72 h, and (d) 96 h using different CUR

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concentrations (50, 100, and 200 μg mL-1). The results showed significant levels (p <

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0.05) when compared to the control (cells treated with the MBN). Figure 10. Cell viability of the astrocyte cells treated with free CUR and the βCD/CUR@MBN at (a) 24 h, (b) 48 h, (c) 72 h, and (d) 96 h using different CUR concentrations (50, 100, and 200 μg mL-1).

Tables Table 1. Parameters calculated from the Korsmeyer-Peppas model and kinetics models used to fit the CUR release data. Model

Parameter -2

Korsmeyer-Peppas

Determined value

𝑘𝐾𝑃 (x 10 )

2.690

𝑛

0.960

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Journal Pre-proof

First-order

Second-order

R2

0.992

𝑘1 (x 10-2 h-1)

6.800

R2

0.963

𝑘2 (x 10-3 mg h-1)

3.380

R2

0.979

Author Contribution Statement

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Matheus S. Gularte: Methodology, Formal analysis, Investigation Rafael F. N. Quadrado: Conceptualization, Methodology, Investigation, Writing -

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Original Draft

Nathalia S. Pedra: Methodology, Formal analysis, Investigation

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Mayara S. P. Soares: Methodology, Formal analysis, Investigation

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Natália P. Bona: Methodology, Formal analysis, Investigation Roselia M. Spanevello: Supervision, Writing - Review & Editing

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Highlights

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André R. Fajardo: Supervision, Project Administration, Writing - Review & Editing

> A polymeric membrane of cationic starch and poly(vinyl alcohol) was prepared; >A β-cyclodextrin/curcumin inclusion complex was efficiently embedded into the membrane; >The complex-loaded membrane allows to control and sustain the curcumin release process; >The complex-loaded membrane exhibited enhanced cytotoxic effect against cancer cells; >The prepared material was non-toxicity against normal cells.

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10