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Journal of Microencapsulation Micro and Nano Carriers

ISSN: 0265-2048 (Print) 1464-5246 (Online) Journal homepage: http://www.tandfonline.com/loi/imnc20

Poly (lactic-co-glycolic acid) nanoparticles for sustained release of allyl isothiocyanate: Characterization, in vitro release and biological activity David Encinas-Basurto, Jaime Ibarra, Josué Juarez, María G Burboa, Silvia Barbosa, Pablo Taboada, R Troncoso-Rojas & Miguel A Valdez To cite this article: David Encinas-Basurto, Jaime Ibarra, Josué Juarez, María G Burboa, Silvia Barbosa, Pablo Taboada, R Troncoso-Rojas & Miguel A Valdez (2017): Poly (lactic-co-glycolic acid) nanoparticles for sustained release of allyl isothiocyanate: Characterization, in vitro release and biological activity, Journal of Microencapsulation, DOI: 10.1080/02652048.2017.1323037 To link to this article: http://dx.doi.org/10.1080/02652048.2017.1323037

Accepted author version posted online: 28 Apr 2017.

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Date: 01 May 2017, At: 07:40

Poly (lactic-co-glycolic acid) nanoparticles for sustained release of allyl isothiocyanate: Characterization, in vitro release and biological activity David Encinas-Basurto1, Jaime Ibarra1, Josué Juarez1, María G Burboa2, Silvia Barbosa3, Pablo Taboada3 Troncoso-Rojas R 4 and Miguel A Valdez1

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1. Departamento de Física, Posgrado en Nanotecnología, Universidad de Sonora, Rosales y Transversal, 83000 Hermosillo, Sonora, México 2. Departamento. de Investigaciones Científicas y Tecnológicas, Universidad de Sonora, Rosales y transversal, 83000, Hermosillo, Sonora, México 3. Departamento de Física de la Materia Condensada, Facultad de Física, Universidad de Santiago de Compostela, España 4. Coordinación de Alimentos de Origen Vegetal, Centro de Investigación en Alimentación y Desarrollo, A.C. (CIAD, AC), Carretera a la Victoria Km 0.6, La Victoria. Hermosillo, Sonora 83000

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E-mail: mail to:[email protected]; cell number: 6621400372; Departamento de Física, Posgrado en Nanotecnología, Universidad de Sonora, Rosales y Transversal, 83000 Hermosillo, Sonora, México

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Highlights

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Low solubility and nucleophilic degradation are main limitations of isothiocyanate compounds.

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Nanoencapsulation of natural compounds could be a promising approach for protect from degradation and enhance antiproliferative properties.

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Biopolymer nanoparticles are recent natural compounds delivery systems.

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Graphical abstract

Abstract The objective of this study is to establish the ability of entrap allyl isothiocyanate (AITC) into polymeric nanoparticles to extend its shelf life and enhance its antiproliferative properties. Natural compounds, such as AITC , have showed multi-targeting activity resulting in a widerange spectrum of therapeutic properties in chronic and degenerative diseases, conversely with most current pharmaceutical drugs showing single targeting activity and often result in drug resistance after extended administration periods. In these sense, AITC -loaded poly (lactic-co-

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glycolic acid) nanoparticles (PLGA NPs) reduced AITC degradation and volatility and were able

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to extend AITC shelf-life comparing with free AITC (65 % vs 20 % in 24 hours respectively). Cell viability and uptake of AITC-loaded nanoparticles were studied in vitro, showing that the

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protection and sustained release of AITC from polymeric NPs involved a larger toxicity of tumoral cells. These nanoparticles could be used as protective systems for enhance biological

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activity.

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Keywords: Allyl isothiocyanate, stability, PLGA, nanoparticles, cytotoxicity

Introduction Cancer is the second leading cause of death worldwide and is expected to surpass heart diseases as the principal one in the next few years 1. Cancer development can be identified by specific hallmarks, including uncontrolled division of cells, sustained proliferative signaling, loss of growth suppressor functions, enhanced angiogenesis and/or invasion of tumoral cells of primarily non-affected organs/tissues (metastasis) 2. Current cancer treatments focused on a single therapeutic modality (chemotherapy,

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radiotherapy, or surgery, for example) are of limited success in many cases due to the

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impossibility of complete tumoral resection, the development of chemo/radio

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resistances, the non-specific systemic distribution of antitumor agents, the achievement of sub-optimal drug concentrations reaching the tumor site, and the generation of 3-4

. Several natural compounds have been long

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adverse side effects and/or cytotoxicity

used in medicine as a consequence of their potential properties as chemo agents in 5-7

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several chronic diseases

. The advantage of these compounds are their multi-

targeting activity resulting in a wide-range spectrum of antimicrobial, antioxidant,

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proapoptotic, antiproliferative, antiangiogenic and enzyme modulation activity properties that are unregulated in tumoral cells

8-11

. Conversely, most current pharmaceutical

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drugs have single targeting activity and often result in drug resistance after extended administration periods; in addition, natural compounds are generally less cytotoxic and less costly than synthetic drugs thanks to a general wider availability from their natural sources under relatively easy extraction and purification processes 2, 12. Isothiocyanates (ITC) are natural compounds produced by certain plants and vegetables of the Cruciferae family that have selective and specific biological activities

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against carcinogenesis and cancer development

. In particular, allyl isothiocyanate

(AITC) is one of the most common naturally occurring ITCs

14

. This compound can stop

tumor cell proliferation by inhibiting carcinogen activation, promoting carcinogen detoxification, inducing cell cycle arrest, activating apoptosis and/or inhibiting cancer cell invasion and metastasis as observed in several types of cancers such as bladder, brain malignant glioma, cervical and colorectal ones

15-17

. These outstanding properties

hence potentially offer the possibility of using ITC in cancer therapy. However, the

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biomedical application of this active compound has been precluded by its poor water

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solubility, volatility, and strong electrophile behavior, which makes it susceptible of nucleophilic attacks of molecules bearing –SH, NH2, and specially, H2O and OH 18-20

. Previous studies (Chen and Ho

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, Ohta, et al.

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moieties

22

) have analyzed the

degradation pathways of AITC in aqueous solution, being N,N-diallylthiourea the major 23

also studied the stability of AITC at different pH

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degradation product. Pechacek, et al.

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(4, 6 and 9) observing that the slowest degradation of AITC was achieved under acidic conditions. In addition, although AITC is efficiently absorbed in vivo and its oral

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bioavailability is above 80% this molecule forms conjugates with biomolecules, especially proteins and enzymes as glutathione

19, 24

, hence, losing/compromising its

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potential therapeutic activity.

A promising strategy to overcome the former limitations is to encapsulate AITC into polymeric nanoparticles to protect them from degradation in the complex physiological media, while achieving a controlled and sustained drug release, hence favoring an increase in its blood half-life, and promoting its accumulation in the site of action either by passive or active mechanisms to enhance its antiproliferative cell properties

25-27

. In

this regard, very few studies have been conducted to protect AITC from the biological environment, and these were mostly related to their potential antimicrobial properties for food industrial applications using matrix hosts as β-ciclodextrins

28-29

, poly (lactic acid)

films 30, arabic gum 31-32 and nanoemulsions 33. Poly-D,L-lactide-co-glycolide (PLGA) is a FDA-approved biodegradable copolymer ideally suited for the development of nano- and micro-particle-based drug delivery systems and scaffolds for tissue engineering

34-35

as a consequence of their

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biodegradability inside the body in biocompatible metabolite monomers, lactic acid and

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glycolic acid, and their commercial availability in a wide range of molecular weights and compositions. For these reasons, PLGA nanoparticles (PLGA NPs) have been used for

nucleic acids, amongst others)

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the encapsulation of a wide range of active compounds (drugs, vaccines, proteins, or which exhibit high stability and loading capacities, and

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offer various feasible routes of administration. Hence, in the present study we obtained

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and characterized AITC-loaded PLGA NPs by a simple emulsion-solvent evaporation using polyvinyl alcohol (PVA) as a stabilizer with the aim of investigating whether PLGA

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NPs are able to protect this natural compound against degradation in aqueous environments and improve their anti-cancer properties, hence, becoming in a potential

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nanoparticulate-based drug delivery vehicle with strong antiproliferative properties for tumoral cells. In vitro tests using cancerous HeLa and MDA-MB-231 cells, as well as retinal epithelial ARPE-19 cell line, allow us to elucidate that drug-loaded PLGA NPs internalize to greater extents and exert a more cytototoxic activity in malignant cells than free AITC, without notorial harmful effects to healthy ones, releasing the therapeutic cargo in a sustained manner.

EXPERIMENTAL SECTION Materials AITC (95% GC purity) was purchased from Fluka Chemical Co. (Steinheim, Germany). Poly-D,L-lactide-co-glycolide (PLGA) 50:50 with molecular weight 30,000–60,000 Da, acetone (99%), fluorescein isothiocyanate (FITC), and PVA were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). HeLa, MDA-MB-231 and ARPE-19 cells were from Cell Biolabs (San Diego, CA, USA). ProLong® Gold antifade reagent DAPI,

Dulbecco’s

modified

eagle

medium,

fetal

bovine

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with

serum

(FBS,

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penicillin/streptomycin, sodium pyruvate, and MEM non-essential amino acids (NEAA) were purchased from Invitrogen (Carlsbad,USA). Dialysis membrane tubing (molecular

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weight cutoff ∼3500) was purchased from Spectrum Laboratories, Inc. (Rancho

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Dominguez, California).

Preparation of AITC-loaded PLGA Nanoparticles AITC-loaded PLGA nanoparticles were obtained by using a simple emulsion-solvent

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evaporation method as described previously, with some modifications

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. Firstly, an

organic phase was prepared by dissolving different amounts of AITC (1-10 mgml-1) with

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6 mg of PLGA in 0.6 ml of acetone to optimize drug loading and encapsulation efficiency. Subsequently, the organic phase was added dropwise to an aqueous one under moderate stirring containing 3% of polyvinyl alcohol (PVA) as an emulsifier to form an oil/water-type emulsion. The resulting emulsion was maintained under gentle stirring for 4 h until complete evaporation of acetone. Next, polymeric NPs were collected by centrifugation at 13500 rpm for 30 min, washed twice with deionized water

and resuspended in 1 ml PBS buffer (pH 7.4) for further analysis. The amount of drug entrapped inside of NPs was determined by Uv-Vis spectroscopy using a calibration curve (R2=0.99) of AITC in acetone at λmax of 242 nm (0.1, 0.5, 1, 1.5, 2, 2.5 mgml-1 of AITC). Briefly, after centrifugation the precipitated drug-loaded NPs were dissolved in acetone for 10 minutes to disrupt the PLGA NPs, releasing the AITC to the bulk acetone solution for all concentrations. The drug-loading content (DLC %) and the drug-

DLC% = (Weight of Drug in NPs/Weight of NPs) × 100

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entrapment efficiency (EE %) were calculated as:

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EE% = (Actual AITC Content/Theoretical AITC Content) × 100

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Empty PLGA NP’s were used as control following the same procedure described above.

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Particle size and morphology characterization

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After resuspending the NPs in 1 ml of PBS buffer, particle size, size distributions and zeta potentials were analyzed using a Zetasizer-Nano ZS (Malvern instruments, UK). The morphological characterization was conducted using both scanning electron

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microscopy (FESEM Ultra Plus, Zeiss, Germany) and atomic force microscopy (AFM, model JSPM-4210, JEOL, Japan). SEM samples were prepared for analysis by

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evaporating a drop of the NP dispersion on a silicon wafer followed by coating for 20 s with Iridium alloy at 20 mA. For AFM measurements, a drop of NPs suspension was deposited onto freshly cleaved mica and observed in non-contact mode using a NSC15 silicon cantilever (MikroMasch, Oregon, USA). The resulting AFM images were analyzed with WSxM software.

Determination of AITC entrapped inside polymeric NPs The amount of AITC encapsulated within the NPs was determined by HPLC analysis

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.

NPs solution was centrifuged and 50 ul of supernatant were injected into HPLC in order to determine encapsulation efficiency . Chromatographic analyses were performed with an HPLC 1200 system (Agilent Technologies, Santa Clara, CA) using a Phenomenex Luna C 18 (30 mm x 2 mm, 5 µm particle size) reversed-phase column. The column was thermostated at 25°C and the analysis was performed in isocratic mode (water and

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acetonitrile, 35/65 (v/v)) at 1 ml min-1 flow rate for 10 min. Quantifications were carried

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out at 242 nm using an external calibration curve. The DLC % and EE % were

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calculated as mentioned before.

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Stability of Allyl isothiocyanate in PLGA NPs

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NPs dispersions were extracted with organic solvent at physiological pH and degradation products were analyzed by HPLC. AITC-loaded NPs and free AITC aqueous solution (containing 0.1 %(v/v) DMSO) were placed in a water bath at 37 °C.

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Next, 0.5 ml of both samples were taken at different time intervals (0, 24, 48, 72, 96 and 120 h), vortexed with 0.5 ml of acetonitrile and centrifuged at 9000 x g for 30 min. All

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mixtures were passed through a 0.2 µm membrane filter before being injected into the HPLC

equipment

with

same

condition

describe

above.

HPLC-ESI-MS/MS

measurements were performed using an Agilent Technologies 6330 Ion Trap MS coupled to an Agilent 1200 HPLC. A PEEK tubing (30 cm × 0.1 mm (i.d.)) was used to connect directly the ion-exchange HPLC column to the electrospray source. Data

analysis was performed using Bruker Daltonik Data Analysis for 6300 Series Ion Trap LC/MS.

AITC release from PLGA NPs Drug release profiles in acidic and neutral pH conditions were obtained by dialysis under sink conditions. Drug-loaded polymeric NPs were suspended in 1 ml PBS solution and placed in dialysis membranes (Spectre, 3500 Da MW). The membranes

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were soaked in 20 ml of phosphate buffer saline solutions at pH 5.0 and 7.4, and

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maintained at 37 ºC under stirring at 100 rpm. At predetermined time intervals (0-150 h),

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individual aliquots of 1 ml were taken out to quantify the amount of released drug by

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HPLC, replacing the same volume with fresh phosphate buffer saline solution.

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Cell culture and Cell viability by MTT assay HeLa, MDA-MB-231 and ARPE cell lines were obtained from Cell Biolabs and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% (v/v) fetal

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bovine serum, 100 IU ml-1 of penicillin and 100 μg ml-1 of streptomycin and 1% non-

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essential amino acids. Cells were maintained in an incubator supplied with a 5% CO2/95% air-humidified atmosphere at 37 °C. Cell lines were placed in 96-well plates at a density of 104 cells per well and incubated for 24 h to allow cell attachment

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. Cells

were then incubated for 48 h with bare NPs (polymer concentration: 6, 3, 1.5, 0.75, 0.37 and 0.18 mgml-1), AITC-loaded NPs (AITC concentrations of 0.65, 0.32, 0.16, 0.08, 0.04 and 0.02 mgml-1) and free AITC at the same concentrations as controls in order to determine the IC50. After incubation, NP-containing medium was replaced with DMEM

containing 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) (5 mgml1

), and cells were additionally incubated for 4 h. Then, 150 µl of isopropanol were added

to dissolve the formed formazan crystals. Absorbance was measured at 530 nm using a BioRad microplate reader. Untreated cells were taken as negative control. Experiments were carried out in triplicate and results expressed as mean values ± standard deviation (n=3)

polymeric

NPs

were

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Fluorescent-labeled

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In vitro cellular uptake of nanoparticles prepared

by

Fischer

esterification

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incorporating FITC to PVA before emulsification. Briefly, a 3% (w/v) PVA solution was acidified by adding sulfuric acid up to pH 3.5; then, 30 g of FITC were added to a 3%

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(w/v) PVA solution, left under magnetic agitation for 12 h and dialyzed for 24 h to

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remove unbounded FITC. Confirmation of ester bond formation was made using a FTIR spectrometer equipped with an universal ATR accessory. To test the successful incorporation of drug-loaded NPs into cells, 6-well plates were placed with HeLa and

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MDA-MB-231 cells at a density of 105 cells per well and incubated at 37 °C for 24 h to allow cell attachment. Afterwards, the medium was replaced by fresh one containing

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FITC labeled-NPs at a concentration of 2 mgml-1 for 4 h. After incubation, NPs were removed and 2-3 washes were performed, and cells subsequently fixed with 4% (w/v) paraformaldehyde in PBS, washed and stained with BODIPY Phalloidin in 0.2% (w/v) Triton X-100 and stained with one drop of ProLong Gold antifade DAPI reagent. The cellular uptake was analyzed using confocal laser scanning microscopy (Leica TCS

SP5, Germany) under different excitations (DAPI (blue-coloured, exc = 355 nm), BODYPI (red-coloured, exc = 503 nm); and FITC (green-coloured, exc = 480 nm).

3. Results and Discussion

3.1 AITC-loaded PLGA NPs

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In this study we obtained stable PLGA NPs through an emulsion-solvent evaporation

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method in order to allow the encapsulation of AITC to great extents while protecting it from its degradation in the surrounding aqueous environment. To optimize the

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maximum amount of AITC that can be encapsulated, we evaluated different initial feeding drug concentrations (1 to 10 mg ml-1) for loading. For the lowest concentration

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(1 mg ml-1) an encapsulation efficiency of ca. 81.0% was reached, with ca. 0.8 mg ml-1

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AITC entrapped within the NPs. As the initial feeding drug concentration increases, the amount of loaded drug increases, but the encapsulation efficiency progressively

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decreases up to 10 mg ml-1 of initial fed drug, for which ca. 2.8 mg ml-1 AITC were encapsulated while decreasing EE to 29.0%. This effect points to a progressive NP

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saturation (Table 1). On the basis of these data and in order to have a suitable balance among the amount of loaded drug and drug loading efficiency, we chose an initial AITC concentration of 4 mgml-1 to be used in subsequent experiments.

3.2 Particle size, surface charge and morphology characterization Despite structurally-related AITC compounds with rather similar physico-chemical properties have been previously encapsulated in PLGA as antimicrobials in food preservation as mentioned previously, to our best knowledge this is the first time that AITC has been encapsulated into PLGA NPs for its sustained controlled release for chemotherapeutical applications. Firstly, particle sizes of PLGA, PVA-stabilized PLGA (PLGA/PVA) and AITC-loaded PLGA/PVA NPs were observed to be ca. 150 ± 3, 196 ±

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5.5 and 200 ± 3 nm, respectively, with a polydispersity > 2%, and within the limits for

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successful administration via parenteral route. Also, ζ-potentials of -30.7 ± 1.5 for bare PLGA, -0.8 ± 0.2 for PLGA/PVA and -8.0 ± 0.9 for AITC-loaded NPs were obtained, 40

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respectively, in agreement with previous reports

. The drastic reduction of zeta

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potential for PLGA/PVA NPs confirmed the presence of PVA chains shielding the 41

. It might be also

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negatively charged -COO- moieties of PLGA at NPs surfaces

possible that some AITC molecules trapped inside the polymeric NPs during the synthesis can interact with free functional COOH- and OH groups on their surface as a

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consequence of the electrophilic nature of isothiocyanate (N=C=S)

22-23, 42

, as reflected

from the differences in zeta potentials observed betweem PVA and AITC-loaded-

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PLGA/PVA NPs. Nevertheless, despite the low zeta potential values of PLGA/PVAstabilized NPs, the particles were stable at least for several weeks, because of that the PVA polymer provides a successful steric stability. On the other hand, NP sizes and morphologies were further evaluated by AFM and SEM microscopies. NPs appeared spherical in shape, and with sizes smaller than those

obtained by DLS (ca. 150 nm instead of ca. 200 nm) probably as a result of shrinking of polymeric chains upon drying for microscopy visualization (Figure 1)

3.3 Stability of AITC AITC easily degrades in aqueous media due to nucleophilic attacks, and/or pH and temperature variations. In order to know the extent of degradation of this active

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principle, AITC-loaded PLGA NP´s were subjected to HPLC analysis in order to monitor

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the possible degradation products of AITC at room temperature. Chromatograms observed in Figure 2 shows the peak corresponding to pure AITC (retention time: 6.7

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min) at different concentrations, whilst that corresponding to the supernatant is shown in Figure 2b. In this Figure, a new peak at a retention time of ca. 3 min is clearly observed, Several studies have

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which should correspond to a degradation product of AITC.

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shown that AITC decomposition by nucleophilic attacks of OH groups in water can result in different metabolites

19, 21-23

. For example, Pechacek, et al.

23

evaluated the

decomposition products of AITC in aqueous solutions at different pH (4, 6 and 8)

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observing that the decomposition rate occurred slowly under acidic conditions. The major degradation products found were allylamine and carbon disulfide in alkaline and

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acidic media, respectively. Also, Kawakashi and Namiki found that the main decomposition products in aqueous medium at 37 ºC were diallylthiourea, allyl allyldithiocarbamate, diallyl tetrasulfide and diallyl pentasulfide. Figure 3a confirms that encapsulated AITC inside NPs (2.3 mgml-1) does not show any sign of degradation at 0 h of incubation whilst after long incubation some degradation takes place as observed by the presence of the retention peak at ca. 3 min in agreement with the observations

corresponding for free AITC after 5 days of incubation at 37 °C (Figure 3b). This suggests the appearance of a new chemical form, which is more hydrophilic than AITC (it eluted earlier), in contrast with AITC-loaded PLGA NP's, greater AITC remained without degradation after 5 days (Figure 3c). Other small peaks appearing in AITCloaded NPs after short and extended incubation might be related to degradation of PLGA polymer. Electrospray ionization mass spectrometry of the fraction collected a 3 min by HPLC spectra indicated that the major degradation products found were

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allylamine, allyl mercaptan as reaction intermediates, and diallylurea and diallylthiourea

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as main final decomposition products, in agreement with a decomposition reaction in midly alkaline medium (Figure S1). The presence of allylamine and allylmercaptan

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would also point to the decomposition reaction is not complete. On the other hand, an evaluation of the amount of active and unreacted AITC inside polymeric NPs show that,

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for example, after 24 and 120 h of incubation at 37 °C, nearly 75 and 21% of AITC was

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detected, respectively, within the NPs. In contrast, only 20 and 1% of original free AITC remained in suspension in water after these periods of time, respectively (Figure 3d). In

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this way, PLGA NPs clearly slower drug degradation and protect AITC to larger extent

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than the free drug, enabling a superior biological activity of the cargo compound.

3.4 Release of AITC from PLGA NPs The cumulative release of AITC-loaded PLGA NPs was investigated under different solution conditions, particularly those mimicking the cell cytosol (7.4) and cell lysosomes (5.0) pH for 150 h at 37 C (Figure 4). We observed that the release profiles were rather similar under both solution conditions, that is, an initial burst release phase was present followed by a slower and sustained release one. The fast release at the first 3 h could

be originated from the presence of adsorbed AITC molecules on the polymeric NP´s surface during the particle formation process; next, the release began to be slower and more sustained. Under acidic conditions, AITC-loaded NPs showed a faster release (ca. 50 %) if compared with neutral pH (ca. 36%) during the first 24 h of incubation. At longer incubation times, this trend is maintained, with cumulative releases of up to 86% and 56 % for acidic and neutral pH, respectively. It is worth mentioning that drug release from PLGA NPs generally takes place via polymer erosion and diffusion (Faisant, et al. 43, the

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former taking place via hydrolysis of the ester bonds in the polymer backbone. It is

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widely established that PLGA degradation starts with water uptake, with hydrolysis leading to the production of acidic oligomers. Rather similar fast release profiles have

al.

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been also observed for AITC encapsulated inside other matrices. For example, Kim, et entrapped AITC in calcium alginate gel beads and AITC released in simulated

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gastric fluid (pH 3.2) was almost ca. 60 % after 12 h of incubation for the different gel

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compositions evaluated. Zhang, et al.

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used gelatin–gum arabic coacervate

microspheres for the sustained release of AITC, with also ca. 60 % of the drug releases

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after 48 h under acidic conditions 45-46.

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3.5 Cell viability

The antiproliferative effect of AITC-loaded PLGA NPs on cancerous HeLa and MDAMB-231 cells and retinal epithelial ARPE-19 was assessed by MTT assay. Figure 5a-b show that both HeLa and MDA-MB-231 cancer cells significantly reduced their proliferation after 48 h of incubation in the presence of AITC-loaded NPs to larger extents than in the presence of free AITC. Also, cell viabilities in the presence of AITC-

loaded PLGA NPs was concentration-dependent, with IC50 values of 0.33 ± 0.05 and 0.38 ± 0.1 mg ml-1 in HeLa and MDA-MB-231 cells, respectively, and viability extents of ca. to 25 ± 11% and for Hela and 24 ± 7% or MDA-MB-231 at the highest AITC encapsulation concentration (0.65 mg ml-1), conversely comparing free AITC at the highest concentration (eq. AITC-loaded NP´s) only 33 % ± 8 and 39 % ± 9.of cell viability were reduced for Hela and MDA-MB-231 respectively. These results are in agreement with previous data

15, 47-49

noting the need of relatively high IC50 values of

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free AITC to exert its cytotoxic function as a consequence of the loss of its therapeutic

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activity due to degradation by nucleophilic reactions in aqueous environments

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, and

the lack of sufficient drug accumulation in the targeted cells and the absence of a 50-52

. On the other hand, the survival of endothelial

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sustained release profile within cells

ARPE-19 cell was minimally affected by AITC-loaded NPs (see Figure 5c) even at AITC

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concentrations were above IC50 in both cancer cell lines: For example, 80 ± 11.5 % of

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ARPE cells were viable after a 48 h exposure to 0.32 mgml-1 AITC-loaded NPs, whereas only 53 ± 12 % of HeLa and 56 ± 13% for MDA-MB-231 cells survived at the

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same free AITC concentration. IC50 value of AITC-loaded PLGA NPs in normal ARPE cells was 0.92 ± 0.18 mgml-1, approximately three times higher than in human MDA-MB-

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231 and HeLa cancer cells. These data point to the high and specific cytotoxicity of loaded AITC to cancer cells rather than human epithelial ones Xiao, et al.

47

47, 53-55

. In this regard,

analyzed the cytotoxic effect of AITC against human prostate cancer and

normal cells showing that this natural compound significantly inhibits the proliferation of cultured PC-3 (androgen-independent) and LNCaP (androgen-dependent) human

prostate cancer cells in a dose-dependent manner,. Conversely, survival of a normal prostate epithelial cell line (PrEC) was minimally affected by AITC.

3.7 Cellular uptake of PLGA nanoparticles To analyze the cellular uptake of AITC-loaded PLGA NPs, PVA was previously conjugated with FITC, in order to produce fluorescent PLGA NPs after stabilization with

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the modified stabilizing polymer. Figure S2 shows the FTIR spectrum of PVA

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modification with the dye though linkage of COOH- moiety of FITC with OH- groups in PVA. Main FTIR bands confirm the ester bond formation as carbonyl –C=O stretching

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at 1731 cm-1), in comparison to the band corresponding to the carbonyl group in PVA polymer at ca. 1752 cm-1).

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In order to investigate the cellular uptake of AITC encapsulated inside NPs, HeLa and

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MDA-MB-231 cells were exposed to AITC-loaded polymeric NPs doped with FITC for 4 h. As shown in Figure 6, detectable green fluorescence was observed in the cytoplasm of breast MDA-MB-231 cells upon 4 h incubation with the fluorescent polymeric NPs,

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with part of the polymeric NPs located at the perinuclear region, but without evidence of entrance within the nucleus. To confirm that PLGA NPs are not only absorbed on the

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membrane cell and have been effectively taken up into the cells, the internalization was further studied by performing 3D stacks of perpendicular cross-sectional slices of the samples followed by image reconstruction. Figure 6f shows that NPs are internalized into the cell cytoplasm provided that the green fluorescence from FITC is overlaid with the red fluorescence of the stained cytoplasm, with no obvious fluorescence within the nucleus, as commented above. In the present case, as the polymeric particles are not

functionalized with any targeting ligand, PLGA NPs are nonspecifically endocytosed into the cells probably by fluid phase pinocytosis

56

. Provided that PLGA NPs possess a

slight negative charge, we believe that the cationic sites in the cell membrane should be the main anchorage points to favor interactions for non-specific binding and subsequent cell uptake. Previous works suggested that negatively charged particles bind at the cationic sites in the form of clusters

57-58

because of their repulsive interactions with the

large negatively charge domains of cell surface. In this context, once internalized AITC-

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loaded PLGA NPs might successfully achieve a sustained release of its cargo

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compounds inside the cells enhancing their therapeutic activity.

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4 Conclusions

In this study, the potential use of an encapsulated natural compound, AITC, with

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chemotherapy properties using nanotechnological tools, that is, with its incorporation

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within developed polymeric NPs, to improve its antiproliferative properties of different cancer cell lines without harming normal tissue, was studied. AITC loaded within the

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polymeric particles extend its half-life from degradation in aqueous environments. This also results in a larger cytotoxic effect against tumoral cells as cervical HeLa and breast

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MDA-MB-231 ones.

In addition, AITC activity was found to be superior in these

cancerous cells than in retinal epithelial ARPE-19 ones, as a consequence of the enhanced metabolism of the former. Based on the present results, it can be concluded that polymeric nanoparticle formulations as PLGA NPs encapsulating natural compounds as AITC slow drug degradation by protecting from nucleophilic molecules and volatility, maintaining their therapeutic activity, so they may be considered as an

effective anticancer drug delivery system for cancer chemotherapy without causing harmful effects to normal cells.

ACKNOWLEDGMENTS We thank the Consejo Nacional de Ciencia y Tecnología (CONACyT; México) for financial support through projects CB-2010-01-151794. D. Encinas-Basurto thanks CONACyT for his PhD scholarship. Authors thank Prof. María del Carmen Barciela and

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Prof. Pilar Bermejo for ESI experiments.

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Table 1. Encapsulation efficiency and drug loading of AITC-loaded NPs at different fed initial drug concentration (n=3). AITC-loaded PLGA NP (mg -1 ml )

Encapsulation efficiency (%)

Drug loading (%)

1 2 3 4 5 6 7 8 9 10

0.81 ± 0.15 1.19 ± 0.47 1.77 ± 0.15 2.46 ± 0.29 2.52 ± 0.24 2.55 ± 0.42 2.73 ± 0.26 2.48 ± 0.30 2.58 ± 0.58 2.79 ± 0.40

81.8 59.6 59.2 65.8 47.5 45.6 35.1 32.0 28.6 27.9

11.7 16.7 19.5 24.9 32.1 33.1 35.4 25.8 30.2 35.8

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Theorical AITC -1 (mgml )

FIGURE 1. AFM pictures of a) bare and b) AITC-loaded PLGA NPs and SEM micrographs of c) bare and d) AITC-loaded. PLGA NPs.

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FIGURE 2. a) Chromatogram of standard AITC at different concentrations (0.5, 1, 2, 3 and 5 mg ml-1 ) (retention time: 6.7 min), b) Supernatant of AITC-loaded PLGA nanoparticle after 24 h.

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FIGURE 3. HPLC profiles of AITC and its degradation products during storage at 37 °C. Retention time for AITC was 6.7 min. a) AITC inside PLGA NPs (2.3 mg ml -1) at 0 h; b)

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Free AITC in aqueous solution (initial concentration 2.3 mg ml -1) stored for 5 days at 37 °C; (C) AITC-loaded PLGA NPs (initial concentration (2.3 mg ml-1) after storage for 5 days at 37 °C; d) Percentage of remaining active AITC upon incubation when () encapsulated inside polymeric NPs and () free in aqueous solution (n=3).

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Cumulattive Release %

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60

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5 7.4

140

160

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Time (h) FIGURE 4. In vitro AITC release profile from PLGA NPs at different pH. (Data points represent mean, n = 3).

120

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40 AITC-loaded PLGA NP PLGA NPs Free AITC

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% Viability

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AITC loaded PLGA NPs ¨PLGA NPs AITC

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0 120

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c)

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AITC loaded PLGA NPs PLGA NPs AITC

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0 0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Concentration (mg/ml)

FIGURE 5. In vitro cytotoxicity of free AITC, AITC-loaded PLGA NPs, and bare PLGA NPs at different AITC concentration on a) breast MDA-MB-231, b) cervical HeLa and c) epithelial Arpe-19 cells (n=3).

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FIGURE 6. Confocal microscopy images of AITC-load PLGA NP´s.Nuclei were stained with DAPI (blue channel, exc =355 nm), the actin cytoskeleton with BODYPY Phalloidin (red channel, exc = 633 nm) and PLGA NP labeled with FITC (green channel, exc = ) a) optical microscopy, b) DAPI channel, c) FITC channel, d) BODIPY Phalloidin channel, e) merged image of four channels and f) 3d images of cells incubated with PLGA NP´s. Scale bars 10 µm

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