Polyelectrolyte-coated nanocapsules containing undecylenic acid: Synthesis, biocompatibility and neuroprotective properties

Accepted Manuscript Title: Polyelectrolyte-coated nanocapsules containing undecylenic acid: synthesis, biocompatibility and neuroprotective properties Author: Marek Piotrowski Danuta Jantas Krzysztof Szczepanowicz Sylwia Łukasiewicz Władysław Laso´n Piotr Warszy´nski PII: DOI: Reference:

S0927-7765(15)30064-3 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.07.029 COLSUB 7233

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Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

5-2-2015 28-5-2015 13-7-2015

Please cite this article as: Marek Piotrowski, Danuta Jantas, Krzysztof Szczepanowicz, Sylwia Lukasiewicz, Wladyslaw Laso´n, Piotr Warszy´nski, Polyelectrolyte-coated nanocapsules containing undecylenic acid: synthesis, biocompatibility and neuroprotective properties, Colloids and Surfaces B: Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2015.07.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Polyelectrolyte-coated nanocapsules containing undecylenic acid: synthesis, biocompatibility and neuroprotective properties Marek Piotrowski1, Danuta Jantas2, Krzysztof Szczepanowicz1, Sylwia Łukasiewicz3, Władysław Lasoń2, Piotr Warszyński1 1

Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, 30-239 Kraków, Poland 2

Department of Experimental Neuroendocrinology, Institute of Pharmacology, Polish Academy of Sciences, Smętna 12, 31-343 Kraków, Poland 3

Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland Corresponding author: Marek Piotrowski Postal address: Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, 30-239 Kraków, Poland Phone number: +48126395121 E-mail address: [email protected] Graphical abstract Highlights

Highlights   

Nanocapsules were synthesized using nanoemulsification and the layer-by-layer technique. Biocompatibility of nanocapsules was evaluated in the SH-SY5Y cell culture. Neuroprotective action of nanoencapsulated form of undecylenic acid was evaluated against staurosporine-induced neurodegeneration.

Abstract The main objectives of the present study were to investigate the biocompatibility of polyelectrolyte-coated nanocapsules and to evaluate the neuroprotective action of the nanoencapsulated water-insoluble neuroprotective drug – undecylenic acid (UDA), in vitro. Core-shell nanocapsules were synthesized using nanoemulsification and the layer-by-layer (LbL) technique (by saturation method). The average size of synthesized nanocapsules was around 80 nm and the concentration was 2.5 x 1010 particles/ml. Their zeta potential values ranged from less than -30 mV for the ones with external polyanion layers through -4 mV for the PEG-ylated layers to more than 30 mV for the polycation layers. Biocompatibility of synthesized nanocarriers was evaluated in the SH-SY5Y human neuroblastoma cell line using cell viability/toxicity assays (MTT reduction, LDH release). The results obtained showed that synthesized nanocapsules coated with PLL and PGA (also PEG-ylated) were non-toxic to SHSY5Y cells, therefore they were used as nanocarriers for UDA. Moreover, studies with ROD/FITC-labeled polyelectrolytes demonstrated approximately 20% cellular uptake of synthetized nanocapsules. Further studies showed that nanoencapsulated form of UDA was biocompatible and protected SH-SY5Y cells against the staurosporine-induced damage in lower concentrations than those of the same drug added directly to the culture medium. These data suggest that designed nanocapsules might serve as novel, promising delivery systems for neuroprotective agents. Keywords Nanoencapsulation, polyelectrolytes, biocompatibility, neuroprotection, SH-SY5Y, undecylenic acid Introduction A successful prevention and an effective treatment of the central nervous system disorders such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis represent a major challenge of contemporary medicine [1]. The course of these diseases is also a considerable social problem, affecting not only patients but their whole families [2]. Among various mechanisms of neurodegenerative processes, a disrupted neuronal intracellular calcium homeostasis leading to hyper-activation of μ-calpain could be listed at a very beginning [3,4]. Thus, an inhibition of calpain activity became crucial among approaches in the treatment and prevention of neurodegenerative diseases [5]. However, despite the development of new neuroprotective substances, their effectiveness is still far from satisfactory, mainly due to their chemical constraints (e.g. poor solubility and stability) and pharmacological limitations leading to drug elimination, peripheral toxicity and numerous side effects [6]. Another difficulty is the complexity of the blood–brain barrier (BBB) that hampers the entry of the vast majority of neurotherapeutics and is permeable for small molecular lipophilic compounds only [7,8].

Nanomedicine provides novel strategies to overcome these limitations. Nanoparticulate drug delivery systems (nanocapsules, nanocontainers, etc.) can challenge undesirable properties of the drugs due to their unique physicochemical and pharmacological properties. Comparing to the conventional drugs, such nanosystems may reveal increased bioavailability, diminished toxicity and enhanced cellular uptake. Typically nanocapsules consist of colloidal (e.g. emulsion) core surrounded by a polymeric functionalized shell [9]. There is a number of research on encapsulation of nano- or microemulsion droplets by the sequential adsorption of (bio)polyelectrolytes to form nanocarierrs as drug delivery vehicles for hydrophobic drugs [10-16]. Nanocapsules exhibit similarity in their size and morphology to the natural occurring carriers such as serum lipoproteins [17]. Their diameters in the range between 10 – 200 nm enable them to interact with biological systems at the molecular level [18]. Another relevant feature of nanocapsules is the possibility to provide hydrophobic environment for the encapsulation, protection and delivery of poorly water soluble drugs, e.g. by using emulsion cores. Thus, the use of toxic organic solvents (DMSO, etc.) may be avoided [19]. On the other hand, polymeric shell enables controlling and triggering drug release, what can result in better therapeutic efficacy [20]. Polymeric shell may be further modified, e.g. by PEG-ylation, in order to provide additional steric stabilization in body liquids. Moreover, PEG-ylation prevents nanocapsules from the process of opsonization. Thus, the clearance by the immune system and macrophage uptake may be avoided [21-24]. It is also worth noting, that the most recent data provide evidence that PEG-ylated nanocarriers penetrate BBB and may be suitable for specific delivery of neurotherapeutics into the brain [25]. There are multiple approaches in which colloidal nanovectors can result in improved drug delivery to the central nervous system [26,27]. More and more studies concern the delivery of anticancer drugs, analgesics, anti-Alzheimer's drugs, cardiovascular drugs and several other macromolecules to the brain tissue [10]. These novel strategies show promise for the effective drug delivery in the prevention and treatment of central nervous system disorders [28]. Recently, the medical utility of undecylenic acid (UDA), an 11-carbon unsaturated fatty acid, has been proposed as a promising neuroprotective substance [29]. This suggestion was based on its ability to diminish the cell death induced by amyloid β (Aβ), glutamate and H2O2 in human neuroblastoma SH-SY5Y cells. Among neuroprotective mechanisms responsible for that effect, an inhibition of μ-calpain (a calcium-activated cysteine protease) activity has been postulated [29]. The neuroprotective effects of undecylenic acid in a staurosporineand doxorubicin-induced cytotoxicity model have also been evaluated [30]. It has been observed that UDA decreased the St-induced changes in mitochondrial and cytosolic apoptosis-inducing factor (AIF) level, whereas in Dox-model it affected only the cytosolic AIF content. It has been also observed that an inhibitor of phosphatidylinositide 3-kinase (PI3-K) pathway but not mitogen-activated protein kinase (MAPK) pathway blocked the protection mediated by UDA in those models of SH-SY5Y cell injury [30].Therefore, in present work we decided to synthesize and characterize emulsion-core and polyelectrolyte-shell

nanocapsules designed as a delivery system for undecylenic acid, to investigate their biocompatibility and cellular uptake, and to evaluate their neuroprotective action in vitro. Materials and methods Chemicals Synthetic polyelectrolytes and biopolyelectrolytes (Tab. 1a): PLL (P2636), PAH (283215), PDADMAC (409014), CHI (448869), PGA (P4886), PSS (243051), ALG (180947), and surfactant: AOT – docusate sodium salt (D4422) were purchased from Sigma Aldrich. Chloroform (234431116) was from POCH. UDA – undecylenic acid (W324701), Lissamine rhodamine B sulfonylchloride (86186), Me-PEG-NH2 – methoxypolyethylene glycol amine (06676) and PLL-FITC – fluorescein isothiocyanate-labelled poly-L-lysine (P3069) were from Sigma Aldrich. DMEM – Dulbecco’s modified Eagle medium (41966-029), Trypsin/EDTA solution (25200-056) and FBS – fetal bovine serum (10270-106) were from Gibco. The Cytotoxicity Detection Kit (11644793001) was from Roche Diagnostic. Staurosporine (S4400), MTT – 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (M2128), DMSO – dimethyl sulfoxide (D5879), Triton X-100 (T9284), penicillin and streptomycin mixture (P4458) were from Sigma Aldrich. PBS was from BIOMED. All chemicals were used without further purification. Ultrapure water was obtained using Direct Q UV (Millipore). Nanocapsules’ synthesis Nanocapsules were synthesized using the method described by Szczepanowicz et al. [31] and repeatedly cited in further papers [11,16,32-36]. Briefly, nanocapsules’ synthesis was based on direct encapsulation of emulsion cores in (bio)polyelectrolyte multilayer shells. Emulsion cores were produced by adding surfactant-in-chloroform solution to the aqueous phase containing polyelectrolyte upon gentle mixing. The Food and Drug Administration (FDA)approved, negatively charged oil-soluble surfactant AOT (docusate sodium salt) and the following classes of positively charged polyelectrolytes were used in the core preparation: PLL, PAH, PDADMAC, CHI (Tab. 1a). Obtained capsules cores were stabilized by the surfactant-polyelectrolyte complexes formed at the interface. The optimal surfactant/polyelectrolyte ratio was determined by the zeta potential measurements. It was found optimal when the zeta potential reached the constant value close to the zeta potential of the same polyelectrolyte in solution. Subsequently, the capsules cores were encapsulated in polyelectrolyte multilayer shells. The consecutive layers of polyelectrolytes were adsorbed from their solutions using saturation (without the intermediate rinsing step) layer-by-layer technique [37,38]. Polyelectrolytes used were alternately anionic and cationic, either synthetic or those of biological origin (Tab. 1a). PEG-ylated polyelectrolyte (PGA-gPEG) was synthesized using Me-PEG-NH2 – methoxypolyethylene glycol amine according to Szczepanowicz et al. [16]. Drug-loaded nanocapsules were synthesized as described above except that prior to the emulsification process, model drug (UDA) was dissolved in AOT/HCCl3 mixture. For the synthesis of fluorescently labeled nanocapsules, ROD-PLL or

FITC-PLL was used as the first polycation layer. Rhodamine-labeled polycation (PLL-ROD) was synthesized through the coupling of Lissamine rhodamine B sulfonylchloride derivate to the amine group in PLL according to the protocol [39]. Fluorescein isothiocyanate-labeled polycation (FITC-PLL) was used as purchased. Nanocapsules’ zeta potential determination The zeta potential was measured using LDA (Laser Doppler Electrophoresis). Experiments were carried out using Zetasizer Nano Series (Malvern Instruments). Each value was obtained as an average from three runs of the instrument with at least 20 measurements. The zeta potential of capsules and of polyelectrolytes in solution was measured in 0.015 M NaCl. All measurements were performed at 25 °C. Nanocapsules’ concentration measurements Nanoparticle concentration was determined by NTA (Nanoparticle Tracking Analysis) using NS500 (NanoSight). The concentration of nanocapsules was measured in 0.015 M NaCl. All measurements were performed at 25°C. Nanocapsules’ size analysis The size distribution (hydrodynamic diameter) of nanocapsules was evaluated by DLS (Dynamic Light Scattering). Experiments were carried out using Zetasizer Nano Series (Malvern Instruments). Each value was obtained as average from three runs with at least 10 measurements. The size of the nanocapsules was also measured by NTA using NS500 (NanoSight). All measurements were performed at 25 °C in 0.015 M NaCl. Nanocapsules’ refractive indexes were measured before nanocapsules’s size analysis according to the protocol described in the instruction manual (Abbemat 500, Anton Paar). All measurements were performed at 25 °C. Nanocapsules’ stability studies The stability tests for nanocapsules were based on the time-dependent changes of their size distribution and surface charge. Nanocapsules suspensions were stored at the room temperature for up to 90 days and particle size (hydrodynamic diameter) and zeta potential of nanocapsules were periodically measured using Zetasizer Nano Series (Malvern Instruments). Nanocapsules’ visualization Nanocapsules were visualized using SEM (Scanning Electron Microscopy) (JEOL JSM-7500F). Samples were prepared by pouring nanocapsules suspension in 0.015 M NaCl solution on the cylinders and drying overnight. SH-SY5Y human neuroblastoma cell culture

Human neuroblastoma SH-SY5Y cells were grown in DMEM supplemented with 10 % FBS and 0.1 % penicillin/streptomycin mixture. Cells were maintained at 37 °C in a saturated humidity atmosphere containing 95 % air and 5 % CO2. Cells were counted using LUNATM Automatic Cell Counter (Logos Biosystems, Inc.) and seeded into 96-well plates or 24-well plates with the density of 6 × 105 per ml. 24 h before experiments, culture medium was replaced by DMEM containing 1 % FBS. Nanocapsules’ qualitative cellular uptake Cellular uptake qualification was evaluated analogously to Łukasiewicz et al. [40]. Confocal microscopy was used to analyze the localization of the fluorescently labeled nanocapsules in SH-SY5Y cells. Images were acquired using Leica LSC SP5 laser scanning confocal microscope (Leica) equipped with 63x HCX PL APO NA 1.4 oil immersion lens (Leica). The measurements were performed at 37 °C. Fluorescein diacetate (FDA) was added to the cells in order to visualize the interior of the cells. Rhodamine-labeled nanocapsules (AOT/ROD-PLL/PGA) were incubated with the cells for 2 h. FDA was excited at 488 nm and ROD-PLL at 561 nm. Emission was detected at 500 − 550 and 580 − 650 nm, respectively. Data were registered in a sequential mode. Nanocapsules’ quantitative cellular uptake Accumulation of nanocapsules inside SH-SY5Y cells was measured in a 96-well plate-reader (Infinite M200 PRO, Tecan) similarly to the procedure described by Palama et al. [41]. Cellular uptake of microcapsules was initiated by adding FITC-tagged nanocapsules (AOT/PLL-FITC/PGA/PLL, AOT/PLL-FITC/PGA, AOT/PLL-FITC/PGA-gPEG) to the cells and then incubated at 37 °C for 1-24 h. The experiments were terminated by washing the cells twice with ice cold PBS, after which 0.1 % Triton X-100 in 0.2 M NaOH solution was added to lyse the cells. Cell-associated FITC-labeled nanocapsules were quantified by analyzing the cell lysate in a fluorescence plate reader. FITC was excited at 485 nm, emission was detected at 528 nm. Uptake efficiency was expressed as a percent of the control group, which was the fluorescence intensity of the total amount of FITC-labelled nanocapsules added to the cellfree medium. The FITC-fluorescence data were confirmed and visualized by microphotographs taken during alive imaging of SH-SY5Y cells using Inverted AxioObserevr Microscopy (Carl Zeiss) with fluorescence (480 nm) and Differential Interference Contrast (DIC) methods. Cell treatment Cytotoxicity of empty nanocapsules and neuroprotection of drug-loaded nanocapsules were evaluated on the basis of cellular viability quantification and cell death assessments. For the determination of cytotoxicity of the synthesized cores/nanocapsules (AOT/PAH, AOT/PDADMAC, AOT/PLL, AOT/CHI, AOT/PLL/PSS, AOT/PLL/ALG, AOT/PLL/PGA (1-10 polyelectrolyte layers), and AOT/PLL/PGA-g-PEG), SH-SY5Y cells were treated with 10 μl of particular solutions to achieve the final concentration of ~ 2.5 x 109 particles/ml. The proper

concentration of staurosporine (St, 0.15 μM) for neuroprotective studies were chosen on the basis of our previous work results [42]. For the studies of neuroprotection, UDA with the range of concentrations 0.01 – 20 μM was used. UDA stock solution (10 mM) was prepared in DMSO and was stored at 4 °C. The final dilutions of UDA and St were prepared in distilled water. Nanocapsules were stored and diluted in 0.015 M NaCl. The chemicals were present in cultures at a final concentration of 1 % for St and UDA and 10 % for nanocapsules. Control cells were treated with 10 μl of nanocapsules’ solvent (0.015 M NaCl). Cellular viability quantification Cell viability was quantified using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay as described previously [43]. Briefly, nanocapsules were added to each well (~ 6.3 x 105 particles/cell), and cells were incubated for 24 h at 37 °C. Next, 10 μl of MTT solution (1.5 mg/ml) was added to each well and incubated for 40 min at 37 °C. Subsequently, cells were centrifuged at 1000 rpm for 3 min, and after removing culture medium and excess MTT, purple formazan salt crystals were dissolved in 100 μl DMSO (dimethyl-sulfoxide). After a brief agitation on a plate shaker, the absorbance of each sample was measured spectrophotometrically at a wavelength of 570 nm in a 96-well platereader (Infinite M200 PRO, Tecan). The data were normalized to the absorbance in control cells (control, 100%) and expressed as a mean ± SEM established from n ≥ 5 wells per one experiment from 3 separate experiments. Cell death assessments In order to estimate cell death, the level of lactate dehydrogenase (LDH) released from damaged cells into culture media was quantified as described previously [43]. A colorimetric assay was used, in which the amount of red formazan salt formed after the conversion of lactate to pyruvate, proportional to LDH activity and to the number of damaged cells in the sample, was measured. After 24 h of nanocapsules’ incubation with cells (~ 6.3 x 105 particles/cell) at 37 °C, cell-free culture supernatants (85 μl) were collected from each well and incubated with the reagent mixture according to the manufacturer’s instructions (Cytotoxicity Detection Kit, Roche Diagnostic) for 20 min. The absorbance of each sample was measured at a wavelength of 490 nm using 96-well plate-reader (Infinite M200 PRO, Tecan). Data were normalized to the activity of LDH released from Triton X-100-treated cells (total, 100 %). Results were expressed as a mean ± SEM established from n ≥ 5 wells per one experiment from 3 separate experiments. Statistical analysis Data after normalization to the control or total groups (± SEM) were analyzed using Statistica software (StatSoft Inc.). The analysis of variance (one-way ANOVA) and post-hoc Duncan’s test for multiple comparisons were used to show statistical significance with assumed p < 0.05.

Results and discussion Preparation of nanocapsules In the present work nanocapsules with the core-shell structure were synthesized using nanoemulsification and the layer-by-layer (LbL) saturation method analogous to the method described previously [11,16,32-36,44-46]. Emulsion cores were formed by addition of oil phase to polycation solution during mixing with the magnetic stirrer at 300 rpm. The oil phase for capsules preparation was prepared by dissolution of surfactant AOT in chloroform (330 g/l). Emulsion droplets were formed by adding a fixed 0.1 ml volume of AOT/chloroform to 200 ml of 0.015 M NaCl aqueous polycation solutions in the range of concentrations 5 – 50 ppm under mixing using a magnetic stirrer at 300 rpm. The optimal polyelectrolyte concentration was found when the zeta potential reached the constant value close to the zeta potential of the same polyelectrolyte in solution [11]. As an example, PLL concentration of 40 ppm was used. Then the capsules cores were encapsulated in polyelectrolyte multilayer shells. Subsequent layers of the polyelectrolyte shell were adsorbed on emulsion cores using similar procedure (AOT/PLL/PSS, AOT/PLL/ALG, AOT/PLL/PGA (1-10 polyelectrolyte layers), and AOT/PLL/PGA-gPEG). Fixed volume of positively charged nanoemulsion core was added to the polyanion solutions, and proper polyanion concentration, determined by the zeta potential measyrements, was used in the synthesis process. As an example, volumes of polyelectrolyte (PLL and PGA) of concentration 2000 ppm used to form PLL/PGA layers on capsules core (AOT/PLL; fixed 8 ml volume) are presented in Tab. 1b. Stable nanocapsules were obtained when zeta potential of emulsion core with adsorbed subsequent polyelectrolyte layer reached the constant value close to the value obtained for free polyelectrolyte in solution (Tab. 1c). The observed layer-to-layer variations of zeta potential provide evidence for the formation of consecutive layers. To create PEG-ylated shell, positively charged capsules were coated with a layer of PGA-gPEG using the same LbL procedure, by adding PLL terminated cores into filtered PGA-gPEG solution. Because of the toxicity issue, chloroform was evaporated from suspensions of nanocapsules shortly after preparation of emulsion. The amount of the chloroform after evaporation was determined as not exceeding 0.04 mg/l [40]. Drug-loaded nanocapsules were synthesized as described above except that prior to the emulsification process, model drug (UDA) was dissolved in AOT/HCCl3 mixture to achieve a concentration 74 mg/ml. This concentration corresponds to the 200 µM concentration of the drug in the stock nanoformulation. As undecylenic acid is practically not soluble in water (the logP (P octanol/water partition coefficient) is higher than 3,86 (Material Safety Data Sheet, Sigma Aldrich, W324701) we assumed 100% efficiency of encapsulation. Consequently, without solubility enhancer (such as Polysorbate 80) added to the release media prior to the experiments [15] the released drug concentrations were very difficult to monitor. However, such conditions do not fulfill the in vivo criteria, so that on the basis of such in vitro release experiments the expected in vivo biological effects of the produced nanocapsules cannot be

accurately foreseen. ROD/FITC-labeled nanocapsules were used for quantitative and qualitative cellular uptake studies. The illustrative scheme of synthesized nanocapsules is presented in Fig. 1a. Characterization of nanocapsules As it was concluded on the basis of stable zeta potential and particle size values in time, nanocapsules’ cores consisted of AOT/PLL complexes showed the superior stability stability over AOT/CHI ones. Thus, for further experiments, the emulsion cores stabilized by AOT/PLL complexes were selected. Those cores, coated with polyanion layer PGA or PGA-gPEG were stable up to 90 days [16]. The average size of synthesized AOT/PLL/PGA nanocapsules measured by DLS and NTA was ~ 80 nm (Fig. 1b) and was increasing with the number of adsorbed layers up to ~ 100 nm (for 10 layers). Particle concentration for the final suspension AOT/PLL/PGA, determined by NTA was 2.5 x 1010particles/ml. The examples of SEM micrograph and a sample video frame from NTA of the same AOT/PLL/PGA nanocapsules are shown in Fig. 2. The size of the most of observed particles were below 100 nm (∼ 80 nm), what clearly confirmed the values obtained by DLS. Zeta potential values ranging from below ~ -30 mV for the polyanion layers to above ~ +30 mV for the polycation layers reflect the required surface charge of particles that prevents their aggregation and contributes to their long-term stability in low salt solutions (Fig. 3). The zeta potential of nanocapsules with PGA-gPEG external layer was ~ -4 mV, and were stable (up to 90 days) in physiological conditions due to the steric stabilization mechanisms. In line with biocompatibility, the long-term stability of nanocapsules is another important prerequisite for any pharmaceutical application. Cytotoxicity tests Successful achievements in nanomedicine require detailed understanding nanotoxicology of formulations [47]. Thus, evaluation of cytotoxicity is an obvious first step in assessing the risks associated with applications of nanomaterials in biological systems [48]. Since our main objective is the application of synthesized nanocapsules in neuropharmacology, SH-SY5Y human neuroblastoma cells were chosen. The SH-SY5Y cell line, derived from a bone marrow biopsy of a neuroblastoma patient, has been extensively investigated and widely used in experimental neurological studies as in vitro cell model, including analysis of cytotoxic and neurodegenerative processes [42,49,50]. Divergent cytotoxicity data in the scientific literature convince that results obtained from various biochemical assays may differ depending on the tested agent or the cytotoxicity test employed [51,52]. Therefore, more than one assay should be used to determine cell viability in vitro in order to avoid miscalculation of the results, thus, to increase the reliability of the evaluation of the of the nanocapsules’ cytotoxicity, we decided to perform both MTT reduction and LDH release assays.

The results of the cytotoxicity testing are summarized in Fig. 4. Cell viability and cell death assessments were investigated using MTT and LDH assays, respectively. Average concentration of each nanocpasules tested was ~ 2.5 x 1010 particles/ml what corresponds to ~ 6.3 x 105 particles/cell. Both assays were done 24 h after treatment with the agents. Statistically significant toxic effect was clearly observed for nanocapsules stabilized by AOT/PAH and AOT/PDADMAC complexes (Fig. 4) as it was described before [11]. AOT/PLL and AOT/CHI stabilized cores and cores covered by a second polyanion layer (AOT/PLL/PSS, AOT/PLL/ALG, AOT/PLL/PGA) and PEG-ylated nanocapsules were non-toxic to cells after 24 h of incubation (Fig. 4). The results were comparable when extending the incubation period even to 48 h and 72 h (data not shown). Dilution by half eliminated toxic effects of AOT/PDADMAC and AOT/PAH stabilized nanocapsules’ cores (data not shown). Additionally, we tested cytotoxicity of AOT/(PLL/PGA)n nanocapsules covered by up to ten (n = 5) polyelectrolyte layers (data not shown) and consequently, we did not observe any statistically significant differences with the control group. Those results confirmed that PLL and PGA covered nanoparticles were non-toxic to SH-SY5Y cells, which is in contrast to a common opinion that positively charged surface could be heavily toxic [40]. It appears that the toxicity of nanocapsules in the SH-SY5Y cells is rather related to external layer composition, than to the surface charge. The results obtained in this part are in good agreement with those observed in our previous research where isopropyl myristate (IPM) core and polyelectrolye shell nanocapsules’ cytotoxicity was evaluated on the same cell line [11]. In view of the above, the PLL and PGA covered nanocapsuels were selected for further biological studies. It should be mentioned here that those are widely accepted biodegradable polymers [53,54]; It has been well described in the literature, that PLL and PGA are enzymatically digested [55,56]. Since nanoparticles having large surface areas and chemically active surfaces may sometimes interfere with viability assays, producing false assessments of toxicity and making them difficult to analyze [48], we have measured the extent of particle interference in MTT and LDH tests. The obtained results suggested lack of nanocapsules’ interference on these assays (data not shown). It is also necessary to mention that we did not observe any differences between cells cultured in pure medium and cells treated with 10 μl of 0.015 M NaCl in MTT and LDH tests, thus the impact of the nanocapsules’ solvent on the cytotoxicity results was excluded. Nanocapsules’ cellular uptake Nanocapsules’ cellular uptake refers to the nanocapsules’ ability to penetrate the cell membrane. For quantitative analysis of cellular uptake of nanocapsules we used FITClabeled nanocapsules, which were added to cells cultures for 1, 3 and 24 h. The FITC fluorescence data from cells treated with each type of nanocapsules (AOT/PLL-FITC, AOT/PLL-FITC/PGA, AOT/PLL-FITC/PGA-gPEG) were normalized to the total fluorescence obtained from cell-free system (10 µl of each type of nanocapsules in lysis buffer). We observed a time-dependent increase in FITC fluorescence for each type of FITC-labeled nanocapsules, with relatively higher fluorescence of AOT/PLL-FITC and AOT/PLL-FITC/PGA

nanocapsules (Fig. 5a). As could be noticed on microphotographs (Fig. 5b) the higher intensity of charged nanocapsules is evoked by their aggregation and stickiness to cell membrane. The explanation of those results could be the electrostatic interactions between the nanocapsules and the charged constituents of the culture medium or/and cell membrane. Since AOT/PLL-FITC/PGA-gPEG nanocapsules did not aggregate on the surface of the cells (Fig. 5b), we estimated, on the basis of fluorescence intensity data, that ~ 20 % of each nanocapsules penetrate to the cell interior. Confocal microscopy analysis of the cellular uptake of the fluorescently tagged nanocapsules in SH-SY5Y cells confirms the accumulation of nanocapsules in the cell interior. Representative image illustrating localization of AOT/PLL/PGA nanocapsules in SH-SY5Y cells is presented in Fig. 6. Neuroprotection In order to confirm evidence of the neuroprotective potential of UDA, we tested this compound against the staurosporine (St)-evoked cell damage in human neuroblastoma SHSY5Y cells. We observed about 60 % reduction in cell viability (MTT assay) and increase in cell death (LDH assay) after 24 h of treatment with St (0.15 µM), which was partially prevented by UDA at concentrations 10 and 20 µM, and 20 µM in MTT and LDH tests, respectively (Fig. 7). Control cells in neuroprotection study were always supplemented with the solvent for UDA (1% mixture DMSO/H2O) and this concentration of DMSO (0.5 %) did not influence SHSY5Y cell viability. These results are in agreement with the findings of Lee et al. who observed neuroprotection of 20 µM UDA in the cytotoxicity models using alternative neurotoxins (Aβ, glutamate and H2O2) [29]. Further tests with the nanoencapsulated UDA demonstrated that it significantly attenuated the staurosporine-induced cytotoxicity at 10 times lower UDA concentrations when comparing to the effect of the neuroprotectant alone (Fig. 7). MTT assay revealed that as low concentration as 1 µM UDA in nano-form added to the culture medium exhibits relatively higher protection against St-evoked cell damage than 10 or 20 µM UDA added directly to the culture medium (Fig. 7a). The mechanism of enhanced action of nanoencapsulated drug vs free drug is not yet clear. We suggest that differences in the intracellular metabolism and local concentration of UDA released from nanocapsules and free UDA might account for better efficiency of encapsulated UDA in our model. In LDH assay we demonstrated that already 5 µM of encapsulated UDA exerts similar neuroprotective action comparing to 20 µM UDA alone (Fig. 7b). Taking into account the results of the cellular uptake of FITC- tagged nanocapsules, where we estimated that only ~20 % of AOT/PLL/PGA nanocapsules are transported to the cell interior, we can approximate that the effective neuroprotective concentration of UDA in cells when delivered in the nano-form is much lower when comparing to the effect of UDA added directly to the culture medium, according to both MTT and LDH tests. However, the direct comparison is rather difficult because the percentage of the free drug that is taken up by the cells is difficult to evaluate, as among lipids constituting approximately half of the mass of cell membranes, medium

chained fatty acids are also present. Nevertheless, the encapsulated drug exhibited neuroprotective action in lower doses comparing to the free drug, what means that the neuroprotective action of nanoencapsulated form of UDA is enhanced even at lower than 50 times concentrations. These are important results from the perspective of pharmacology, because the lower dose of the therapeutic drug may reduce its undesirable side effects. Furthermore, it is crucial from an economic standpoint, since the active substances are generally the most expensive part of the drug formulations. Summarizing, by using screening cell viability/toxicity assays in SH-SY5Y cells we demonstrated a higher neuroprotective potential of encapsulated form of UDA, which should be further validated in other cellular and animal models of neurodegeneration. Conclusions Polyelectrolyte coated nanocapsules nanocapsules containing water insoluble undecylenic acid (UDA), that was proposed as a promising neuroprotective substance, were synthesized by direct encapsulation of nanoemulsion core in the polymer shell prepared using the layer by layer (LbL) adsorption. The average size of synthesized nanoemulsions was ~ 80 nm and was increasing with the number of adsorbed layers to ~ 100 nm (for 10 polyelectrolyte layers). PEG-ylated external layer was adsorbed in order to improve biodistribution of nanocarriers. That type of modification should prolong circulation time by elimination of opsonisation process and fast clearance. Biocompatibility of nanocapsules, cellular uptake and neuroprotective action of encapsulated UDA were evaluated on SH-SY5Y human neuroblastoma cells. Cell-viability and cell death assessments (using MTT and LDH assays, respectively) were used to determine biocompatibility/cytotoxicity of the synthesized nanocapsules. The results obtained showed that most of synthesized nanocapsules were non-toxic to SH-SY5Y cells and could be used as drug-loaded nanocarriers, however, for further tests nanocapsules with shell consisting of PLL and PGA biocompatible polyelectrolytes were selected. We showed that synthesized nanocapsules penetrate into SH-SY5Y cells, what was clearly demonstrated by confocal microscopy analysis of encapsulated rhodamine as well as spectrofluorymetrical detection of FITC-labeled nanocpasules. We confirmed that UDA added directly to the culture medium in the range of concentration 10 – 20 µM partially diminished the Staurosporine-induced damage in SHSY5Y cells. Importantly, encapsulated UDA exhibited neuroprotective action in lower doses than the drug added directly to the cell culture medium. The obtained results indicated that encapsulation of neuroprotectants may be considered as novel strategies of inhibition of neurodegenerative processes and the applied methodology may contribute to the further use of nanotechnology in modern therapies. Acknowledgements The work was co-financed by the Interdisciplinary PhD Studies “Molecular sciences for medicine” (co-financed by the European Social Fund within the Human Capital Operational

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1b)

Figure 1 (Panel 1a) Scheme of nanocapsules’ synthesis. (Panel 1b) AOT/PLL/PGA nanocapsules’ size distribution by NTA (red line) and DLS (black line). 2a)

2b)

Figure 2 Sample video frame (Panel 2a) from NTA and SEM micrograph (Panel 2b) of the AOT/PLL/PGA nanocapsules.

Figure 3 Zeta potential distribution of AOT/PLL, AOT/PLL/PGA and AOT/PLL/PGA-gPEG.

4a)

4b)

Figure 4 LDH release (Panel 4a) and MTT reduction (Panel 4b) in SH-SY5Y culture after treatment with hollow shell nanocapsules stabilized by AOT/polyelectrolyte(s) complexes. Each bar represents an average value and SEM taken from n ≥ 5 wells from 3 independent experiments. Comparisons were made using one-way ANOVA followed by Duncan's test (* - p < 0.05, *** - p < 0.001 vs. control group). 5a)

5b)

Figure 5 (Panel 5a) Fluorescence intensity of nanocapsules containing PLL-FITC with the external PLL, PGA and PGA-gPEG polyelectrolyte layer after incubation with SH-SY5Y cells for 1, 3, 24 h and 24 h, respectively (a, b, c - p ≤ 0.001 higher vs. lower time point for each type of nanocapsules; &&& - p ≤ 0.001 and & - p ≤ 0.05 differences between various types of nanocapsules for particular time point (3 or 24 h)). (Panel 5b) Microphotographs of SH-SY5Y cells incubated for 3 h with nanocapules containing PLL-FITC with the external PLL, PGA and PGA-gPEG polyelectrolyte layer.

Figure 6 Confocal microscopy analysis of the localization of the fluorescently tagged nanocapsules (red) in SH-SY5Y cells (green): representative image illustrating localization of AOT/PLL-ROD/PGA nanocapsules in SH-SY5Y cells (left) and vertical scan – along Z-axis (right) of the SH-SY5Y cell after 2 h incubation with nanocapsules.

7b)

Figure 7 MTT reduction (Panel 7a) and LDH release (Panel 7b) in SH-SY5Y culture, after staurosporineinduced cytotoxicity, incubated with undecylenic acid (UDA) added directly to the cell culture medium and the drug added to the medium in the form of AOT/PLL/PGA nanocapsules (e). Neuroprotective effecs has been marked green. Each bar represents an average value and SEM taken from n ≥ 5 wells from 3 independent experiments. Comparisons were made using one-way ANOVA followed by Duncan's test (*** - p < 0.001 vs. control group; # - p < 0.05, ## - p < 0.01, ### - p < 0.001 vs. staurosporine treated cells).

Table 1 (Panel 1a) Characteristics of polyelectrolytes used in the synthesis of nanocapsules. (Panel 1b) Volumes of polyelectrolyte solution (PLL and PGA) used to form PLL/PGA layers of the shell on the capsules core (fixed 8 ml volume). (Panel 1c) Zeta potential values of nanocapsules’ cores stabilized by a complex of AOT and cationic polyelectrolyte and cores stabilized by AOT/PLL and covered by a layer of polyanion or PEG-ylated polyanion. 1a) Polyelectrolyte

Abbrev.

Formula

MW [g/mol]

poly(L-lysine hydrobromide) poly(allylamine hydrochloride) poly(diallyl dimethyl ammonium chloride) poly(D-glucosamine) (chitosan) poly(L-glutamic acid) sodium salt poly(sodium 4-styrenesulfonate) alginic acid sodium salt

PLL

(C6H12N2O2)n · xHBr

PAH

(C3H7N)n · xHCl

30,00070,000 ~15,000

PDADMAC

(C8H16NCl)n

CHI

(C6H11NO4)n

PGA

(C5H9NNaO4)n

PSS

(C8H7NaO3S)n

ALG

(C6H7NaO6)n

Ionic character (zeta potential, 0.015M NaCl) Cationic (+42.7 mV) Cationic (+46 mV) Cationic (+68 mV) Cationic (+38 mV) Anionic (-48 mV) Anionic (-55.7mV) Anionic (-44.7 mV)

100,000200,000 50,000190,000 50,000100,000 ~70,000 120,000190,000

1b) Number of layer

1

2

3

4

5

6

7

8

9

10

Polyelectrolyte PGA used

PLL

PGA

PLL

PGA

PLL

PGA

PLL

PGA

PLL

Volume of PE [ml]

0.2

0.2

0.3

0.3

0.3

0.4

0.4

0.4

0.5

0.2

1c) Emulsion core AOT/PDADMAC AOT/PAH AOT/PLL AOT/CHI

Zeta potential [mV] 62.6 ± 4.89 72.9 ± 5.87 69.5 ± 6.67 38.6 ± 8.01

Nanocapsule AOT/PLL/PSS AOT/PLL/ALG AOT/PLL/PGA AOT/PLL/PGA-gPEG

Zeta potential [mV] -38.5 ± 6.52 -37.8 ± 6.43 -36.2 ± 4.84 -4.33 ± 5.27