Poly-l-asparagine nanocapsules as anticancer drug delivery vehicles

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European Journal of Pharmaceutics and Biopharmaceutics 85 (2013) 481–487

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Poly-L-asparagine nanocapsules as anticancer drug delivery vehicles G.R. Rivera-Rodríguez a,b,1, M.J. Alonso a,b, D. Torres b,⇑ a Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Health Research Institute of Santiago de Compostela (IDIS), University of Santiago de Compostela, Santiago de Compostela, Spain b Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Santiago de Compostela, Santiago de Compostela, Spain

a r t i c l e

i n f o

Article history: Received 14 January 2013 Accepted in revised form 5 August 2013 Available online 13 August 2013 Keywords: Polyasparagine Nanocapsules Nanocarriers Anticancer drug delivery Biopolymers Nanomedicine Targeted therapy Docetaxel

a b s t r a c t This work presents for the first time the development of novel poly-L-asparagine (PASN) nanocapsules and the in vitro evaluation of their potential as anticancer drug delivery systems. The design of PASN nanocapsules was inspired by the well-known avidity of cancer cells for the amino acid L-asparagine together with the expected ability of this hydrophilic polymer to escape to the mononuclear phagocytic system. Besides, these nanocapsules have an oily reservoir, which enables the efficient encapsulation of lipophilic drugs. PASN nanocapsules were obtained by an emulsification-polymer layer deposition process, which involves using a cationic surfactant as a bridge for the interaction of PASN with the lipid core. PASN nanocapsules showed sizes of around 170–200 nm and negative zeta potential values (around 20 mV to 40 mV). The model anticancer drug docetaxel was efficiently encapsulated (around 75%) and retained within the nanocapsule’s structure upon dilution in a simulated physiological medium. Moreover, these nanocapsules exhibited the ability to interact with the NCI-H460 human cancer cells and to enhance the cellular toxicity of the anticancer drug. All these features together with their adequate stability profile render these nanocapsules a new attractive platform for anticancer intracellular drug delivery. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction The development of targeted drug nanocarriers is a revolutionary field of research, which is expected to lead to significant benefits in cancer theranostics [1]. The rational design of these nanocarriers relies on the particular characteristics of the targeted cells and also the tumor microenvironment. Targeting to cancer cells has mainly relied on the identification of over-expressed receptors and the design of the corresponding targeting ligands [2]. Irrespective of the targeting approach, a prerequisite for the targeted nanocarriers is their capacity to evade the mononuclear phagocyte system (MPS) and, thus, to exhibit long-circulating properties. Overall, most of the nanocarriers marketed until now display this capacity [3–5]; however, targeted nanocarriers still remain in the clinical development phase [6]. The general strategy to avoid the rapid plasmatic elimination of nanosystems is the modification of their surface with hydrophilic polymers, such as poly (ethylene glycol) (PEG), poly (amino acids), and polysaccharides. The shield with these hydrophilic polymers ⇑ Corresponding author. Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Santiago de Compostela, Campus Vida, 15782 Santiago de Compostela, Spain. Tel.: +34 881 814 880; fax: +34 981 547 148. E-mail address: [email protected] (D. Torres). 1 Current address: Institute of Plant Biology and Biotechnology, University of Münster, Schlossplatz 8, 48143 Münster, Germany. 0939-6411/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejpb.2013.08.001

enable nanostructures to circulate in the bloodstream for long periods of time and, eventually, to extravasate into the tumor tissues [7]. Among these polymers, the poly (amino acid) poly-L-asparagine (PASN) has been shown to fulfill this objective, with the additional advantage of being biodegraded by lysosomal proteases, thus allowing its complete elimination from the body [8,9]. Furthermore, PASN offers a potential significant advantage for targeting cancer cells. This is related to the fact that the amino acid L-asparagine is an extremely-required nutrient for progression of cancer cells [10,11]. Thus, we have speculated about the dual function of PASN based nanocarriers: long circulation and targeting. Among the variety of biodegradable nanocarriers designed so far for the delivery of anticancer drugs [12–14], our group has particularly focused the attention on biopolymer nanocapsules [15–17]. These are versatile nanocarriers whose core may easily accommodate lipophilic drugs, as most anticancer drugs are, whereas their shell can be conveniently adapted in order to confront specific biological environments and provide the nanocarrier with targeting properties [18]. Based on the above information, the main goal of the current work was to develop a new type of nanocapsules comprising an oily core surrounded by a PASN shell and to assess their potential for the intracellular delivery of anticancer drugs. For the evaluation of this potential, we chose the hydrophobic anticancer drug docetaxel as a model compound.


G.R. Rivera-Rodríguez et al. / European Journal of Pharmaceutics and Biopharmaceutics 85 (2013) 481–487

2. Materials and methods 2.1. Chemicals Docetaxel > 97% (Fluka), Poloxamer 188 (PluronicÒ F68), benzalkonium chloride (BKC), cetrimonium bromide (CTAB), cetylpyridinium chloride (CPC), oleylamine (OLE), trehalose dehydrate, poly-L-asparagine, Hank’s Balanced Solution Salt Serum (HBSS), and Fetal Bovine Serum (FBS) were purchased from Sigma–Aldrich (Spain). MiglyolÒ 812, neutral oil formed by esters of caprylic and capric fatty acids and glycerol, was kindly provided by Peter Cremer Oleo (USA). The surfactant lecithin (EpikuronÒ 145v, a phosphatidylcholine enriched fraction of soybean lecithin) was kindly donated by Cargill (Spain). The N-(fluorescein-5-thiocarbamoyl)1.2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (fluorescein – DHPE), BodipyÒ 558/568 phalloidin and 1,10 -dioctadecyl-3,3,30 ,30 -tetramethyl-indodicarbocyanine perchlorate (DiD) were obtained from Molecular Probes-Invitrogen (USA). RPMI-1640 medium was purchased from ATCC (Spain). 2.2. Construction of PASN nanocapsules Blank PASN nanocapsules were obtained by a modification of the solvent displacement technique previously described by our group [19]. We used two different variations in the method. The first one, named ‘‘one-stage procedure,’’ was based on a dipolar-ionic interaction between PASN, dissolved in an aqueous phase, and the cationic surfactant dissolved in an organic phase, which contains also the oil. Briefly, an organic phase was formed by dissolving 30 mg of lecithin in 0.5 ml of ethanol, followed by the addition of 150 ll of MygliolÒ 812 and 9 ml of acetone containing a cationic surfactant. This organic phase was immediately poured over 20 ml of an aqueous phase composed of the nonionic surfactant poloxamer 188 (0.25% w/v) and the polymer PASN (0.1–1.0 mg/ml). PASN nanocapsules were formed immediately due to the organic solvents diffusion and the PASN interaction with the cationic surfactant at the oil/water interface. Finally, the organic solvents were evaporated under vacuum. The second method applied was a ‘‘two-stage procedure,’’ which involved the incubation of a previously formed cationic nanoemulsion with a PASN aqueous solution. The cationic nanoemulsion was prepared by the method described above but without adding the polymer in the aqueous phase. The phase ratio used in this process was 4:1, v:v (cationic nanoemulsion:PASN solution), and the incubation period was 30 min. The incorporation of the hydrophobic molecules, docetaxel or fluorescent probes, fluorescein-DHPE and DiD, was carried out by adding aliquots of stock ethanol solutions to the organic phase and then following the described procedure. 2.3. Physicochemical characterization of PASN nanocapsules PASN nanocapsules were characterized regarding their size, size polydispersion, zeta potential, and morphology as follows: Particle size and polydispersion index were determined by photon correlation spectroscopy. Samples were diluted to an appropriate concentration with 1 mM of KCl. Each analysis was carried out at 25 °C with an angle detection of 173°. The zeta potential values were calculated from the mean electrophoretic mobility values, which were determined by laser Doppler anemometry. Samples were diluted with KCl 1 mM and placed in the electrophoretic cell. Size and zeta potential evaluations were carried out in triplicate using a NanoZSÒ (Malvern Instruments, Malvern, UK). The nanocapsule morphological determination was carried out by transmission electron microscopy (TEM, CM12 Philips, The

Netherlands). Samples were stained with a 2% w/v phosphotungstic acid solution and placed on cupper grids with FormvarÒ films for analysis. 2.4. Encapsulation of docetaxel into the nanocapsules Docetaxel encapsulation efficiency was determined indirectly, by quantifying the non-encapsulated drug in the formulation. Once the nanocarriers were formed, free docetaxel was removed from the nanocapsules by ultracentrifugation (30,000g, 1 h). This process led to two phases in the sample tubes, a pellet containing the nanocapsules and an aqueous infranatant containing all the free soluble components, including the non-encapsulated docetaxel. Then, analytical samples taken from the infranatant were diluted with acetonitrile and centrifuged for 20 min at 4000g, in order to precipitate any residue of polymers and surfactants. The resultant supernatants were then analyzed by HPLC. Complementary, the total amount of docetaxel into the initial suspension of nanocapsules was estimated. For that, the initial formulation was dissolved in acetonitrile directly and analyzed by HPLC. The HPLC system consisted of an Agilent 1100 Series instrument equipped with a UV detector set at 227 nm and a reverse phase Zorbax EclipseÒ XDB-C8 column (4.6  150 mm i.d., pore size 5 lm, Agilent, USA). The mobile phase consisted of a mixture of acetonitrile and 0.1% v/v orthophosphoric acid (55:45 v/v), and the flow rate was 1 ml/min. The docetaxel quantification was a slight variation in the method described by Lee et al. [20]. The encapsulation efficiency (E.E.) was calculated as E.E. (%) = (A  B)/A  100, where A is the experimental total drug concentration and B is the drug concentration measured in the external aqueous medium, corresponding to unloaded drug. 2.5. Stability of PASN nanocapsules during storage Aliquots of blank nanocapsules suspensions were kept in sealed glass vials at different temperatures (4 and 37 °C). Size and polydispersion index of nanocarriers were monitored at different time points for a period of 3 months; meanwhile, zeta potential values were controlled at the end of the study. Macroscopic aspect (presence of aggregated, cream formation, flocculation, coalescence, changes in color, etc.) was also assessed throughout the period of study. 2.6. Freeze-drying of PASN nanocapsules Blank PASN nanocapsules at different concentrations (0.125%, 0.25%, and 0.5% w/v) were freeze-dried in presence of the cryoprotectant trehalose (0%, 0.25%, 0.5%, and 1% w/v) to evaluate their ability to be transformed in a lyophilized powder. Glass vials were filled with 1 ml of nanocapsules suspension containing the cryoprotectant and then were quickly frozen in liquid nitrogen. The lyophilization consisted in an initial drying step for 60 h at 35 °C, followed by a secondary 24 h drying in a high vacuum atmosphere (Labconco Corp., USA). Finally, the temperature was slowly increased up to 20 °C. PASN nanocapsules were resuspended by adding 1 ml of ultrapure water to the freeze-dried pellet followed by mild agitation and then characterized as explained above. 2.7. In vitro cell uptake and cytotoxicity of docetaxel-loaded nanocapsules 2.7.1. Cytotoxicity (MTT) assay Human non-small lung cancer cell line NCI-H460 was cultured in RPMI-1640 medium (ATCC), supplemented with 10% (v/v) fetal

G.R. Rivera-Rodríguez et al. / European Journal of Pharmaceutics and Biopharmaceutics 85 (2013) 481–487

bovine serum (FBS) at 37 °C in a humidified atmosphere containing 5% carbon dioxide. Tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)2,5 diphenyltetrazoliumbromide (MTT, Acros Organics) was used for mitochondrial activity evaluation. NCI-H460 cells were seeded onto 96-well plates, with a seeding density of 15  103 cells/well in 100 ll culture medium. After 24 h, the medium was removed, and dilutions of docetaxel solution, docetaxel-loaded PASN nanocapsules, and blank PASN nanocapsules in complete medium were added to the wells. Finally, after 48 h of incubation, cell survival was measured by the MTT assay. Briefly, the medium was removed, and the wells were washed twice with 100 ll Hank’s Balanced Salt Serum (HBSS). Then, 20 ll of a MTT solution (5 mg/ml in PBS) and 100 ll HBSS were added to the wells and maintained at 37 °C in an atmosphere with 5% CO2 for 4 h. Afterward, buffers were removed and 100 ll of dimethyl sulfoxide was added to each well and maintained at 37 °C in an atmosphere with 5% CO2 overnight. Absorbance (k = 490 nm) was measured in a BioRad 680 spectrophotometer removing background absorbance (k = 655 nm). The percentage of cell viability was calculated by the absorbance measurements of control growth in the presence of the formulations at various concentration levels. IC50 values were obtained by fitting the data with non-linear regression, with Prism 2.1 software (GraphPad, San Diego, CA). 2.7.2. Cellular uptake studies NCI-H460 cells were seeded in a multiwell-12 plate (Falcon) at 15.7  104 cells/well in supplemented medium for 24 h. Then, the medium was removed, and a volume of 150 ll of the different formulations to be tested was added to each well. These formulations were DiD-labeled PASN nanocapsules, DiD-labeled nanoemulsion, and a dispersion of the fluorescent marker (DiD) in a hydro-alcoholic medium. After a 2 h incubation, cells were washed with acidic phosphate saline buffer (PBS, SIGMA) trypsinized and reconstituted in PBS supplemented with 3% (v/v) of FBS. Living cell suspensions were analyzed for green fluorescence by flow cytometry in a FACScan (Becton Dickinson). On the other hand, confocal microscopy was used to visualize the localization of the fluorescent nanocapsules into the cells. The cells cultured were incubated for 2 h with the treatments. Then, cells were washed three times with cold acidic phosphate buffer (PBS, SIGMA) to eliminate residues of non-internalized nanocapsules. The cells were fixed with paraformaldehyde 4% for 10 min, washed, and counterstained with BodipyÒ phalloidin for 30 min in darkness and refrigerated at 4 °C for 3 h. Finally, after adding a drop of fluorescent mounting medium on the surface of the holders, covers were placed on them for confocal microscope analysis. 2.8. Statistical analysis The statistical evaluation of cell experiments was performed by an ANOVA test followed by a multiple comparison analysis (post hoc Tukey’s test). Differences in other studies were evaluated by means of a t-test for independent samples using Statgraphics Plus XV (Statpoint Technologies Inc., USA). 3. Results and discussion This article describes for the first time the development of a new drug nanocarrier consisting of an oily core surrounded by a poly-L-asparagine hydrophilic shell. The rationale behind the design of this nanocarrier was as follows: the oily reservoir, consisting of Miglyol, is intended to allocate significant amounts of lipophilic drugs, whereas the external poly-L-asparagine (Fig. 1)


Fig. 1. Molecular structure of poly-L-asparagine.

shell is expected to have three differentiated roles: (i) to prolong the plasmatic circulation time after IV administration [8]; (ii) to facilitate the interaction and internalization of the nanocarrier with cancer cells [10]; and (iii) to improve the stability of the drug-loaded oily cores in the biological media and during storage.

3.1. The construction of PASN nanocapsules The principle for the design and construction of PASN nanocapsules was to form oily nanodroplets conveniently stabilized using lecithin and a cationic surfactant. The cationic surfactant was used as a bridge for the subsequent entanglement of a PASN [21]. Preliminary studies were intended to identify the formulation parameters, critical for the formation of stable oily cores and the subsequent deposition of a PASN shell. As a first step, we analyzed the influence of the surfactant type and concentration on the formation of stable oily nanocores. The surfactants used were benzalkonium chloride (BKC), cetrimonium bromide (CTAB), cetylpyridinium chloride (CPC), and oleylamine (OLE), all of them characterized for having cationic amino groups. On the other hand, the surfactant concentrations assayed varied between 0.01% and 0.1%, a range that was defined in order to avoid potential toxicity problems associated with the cationic surfactants. The results showed that the three surfactants assayed allowed the formation of stable cationic nanoemulsions with a size range of 165–244 nm and a cationic surface charge in the range of +30 and +40 mV (Table 1). However, as noted in Table 1, the concentrations required for obtaining stable nanoemulsions varied depending on the type of surfactants. Because of their smaller size, we selected BKC and CTAB at concentrations of 0.04% and 0.02%, respectively, for the subsequent development of these PASN nanocapsules. A second preliminary study was intended to determine the optimal polymer quantity required for the formation of a PASN shell around the nanoemulsions. Thus, selected cationic nanoemulsions were incubated with increasing concentrations of PASN solutions (two-stage procedure, 0.1 up to 1 mg/ml in the final formulation volume). As an example, Fig. 2 shows the evolution of size and surface charge of CTAB-containing nanocapsules as a function of the PASN concentration. It can be noted that upon addition of a low amount of PASN (0.1 mg/ml), a decrease in the zeta potential and an increase in the particle size of the initial cationic nanoemulsion were observed. These changes could be attributed to the polymer deposition around the oily nanodroplets. When the polymer concentration increases within the range of 0.2–0.4 mg/ml, the system aggregated. A low surface charge close to neutrality could explain this phenomenon. However, for polymer concentrations higher


G.R. Rivera-Rodríguez et al. / European Journal of Pharmaceutics and Biopharmaceutics 85 (2013) 481–487

Table 1 Physicochemical characteristics of cationic nanoemulsions prepared with different surfactants: benzalkonium chloride (BKC), cetrimonium bromide (CTAB), cetylpyridinium chloride (CPC), and oleylamine (OLE). Data are the mean values of three replicated batches of each formulation ± S.D. Cationic surfactant

Surfactant concentration (%)

Size (nm)


f Potential (mV)


0.04 0.04 0.02 0.02

208 ± 3 233 ± 10 165 ± 6 244 ± 5

0.1 0.1 0.1 0.2

+33 ± 1 +34 ± 2 +41 ± 3 +39 ± 1

PI: polydispersity index.

Fig. 2. Influence of PASN concentration on the size (dash line) and zeta potential (continuous line) of the nanocapsules prepared according to the two-stage procedure using CTAB as cationic surfactant. Data presented are the mean values of three replicated batches for each experimental condition ± SD.

Table 2 Physicochemical characteristics of blank and docetaxel- or DiD-loaded PASN nanocapsules prepared according to the one-stage procedure (PASN concentration was 1 mg/ml). Formulation

Size (nm)


f Potential (mV)

Enc. Eff. (%)

Blank PASN-CTAB NCs Blank PASN-BKC NCs Docetaxel-loaded PASN-CTAB NCs Docetaxel-loaded PASN-BKC NCs

174 ± 5 162 ± 2 187 ± 3

0.1 0.1 0.1

24 ± 3 20 ± 6 32 ± 8

– – 76.0 ± 3

166 ± 6


21 ± 7

73.0 ± 5

DiD-loaded PASN-CTAB NCs

184 ± 4


34 ± 11

79.5 ± 6

PI: polydispersity index; Enc. Eff.: encapsulation efficiency. Data presented are the mean values of three replicated batches ± SD.

than 0.4 mg/ml, an inversion of surface charge was observed, thus evidencing the effective adsorption of the PASN to surface and the consolidation of the polymeric shell. A further increase in the polymer concentration (beyond 0.6 mg/ml) did not have a significant effect on size or zeta potential values of the nanocapsules. This was probably due to the fact that the surface of the oily cores was saturated in PASN. Similar results obtained for BKC-containing nanocapsules prepared by the two-stage procedure were included as Supplementary material. Based on these results, a concentration of 1 mg/ml which assures the surface saturation by the polymer was chosen for further studies with both surfactants. As mentioned above, based on the physicochemical data, we have hypothesized that the formation of the PASN shell was mediated by the ionic-dipolar interaction between amino groups of cationic surfactant and the polar carboxylic groups of PASN [21]. Taking into account the above information, we proceeded with the formation of nanocapsules according to the one-stage procedure. In this case, PASN was dissolved in the aqueous dispersing phase where the formation of the nanocores takes place. Results of size and surface charge of nanocapsules prepared by the one-stage procedure with both surfactants as a function of the PASN concentration were included as Supplementary material. The physicochemical characteristics of the resulting PASN nanocapsules prepared with a PASN concentration of 1 mg/ml are shown in Table 2. The results indicate that the size of these nanocapsules was significantly smaller than that of the nanocapsules prepared according to the two-stage procedure (254 and 236 nm, for PASN-CTAB and PASN-BKC nanocapsules, respectively), the smallest size being observed for nanocapsules prepared with BKC. This significant size reduction in the nanocapsules could be attributed to a more effective entanglement of the cationic surfactant with the anionic polymer around the oily nanodroplets. The resulting nanocapsules were stable in suspension and exhibited a well-defined spherical and homogeneous spherical shape (Fig. 3). In order to further assess their acceptable pharmaceutical profile, a long-term stability and freeze-drying studies were conducted.

3.2. Stability of nanocapsules during storage The stability is a critical issue in the development of any pharmaceutical formulation. Particularly, variations in temperature during storage are known to compromise the stability of many colloidal systems. In this sense, it is known that surface charge of colloidal systems usually plays a significant role in their stability [22]. The results of the stability study indicated that PASN nanocapsules prepared with CTAB remained stable upon storage at 4 °C and 37 °C

Fig. 3. Transmission electron micrographs of PASN nanocapsules obtained by the one-stage method. Left: PASN nanocapsules prepared with BKC as cationic surfactant; Right: PASN nanocapsules prepared with CTAB as cationic surfactant.

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Fig. 4. Particle size of the reconstituted freeze-dried PASN nanocapsules prepared with CTAB according to the one-stage procedure. Different concentrations (w/v) of nanocapsules were lyophilized using trehalose at 10% (gray bars), 5% (black and white bars), and 2.5% (black bars) w/v concentrations (mean ± S.D.; n = 3).

Fig. 6. Effect on H460 cell viability of PASN nanocapsules prepared with the surfactant CTAB (1.8 mg) after 2 h (A) or 48 h (B) of contact with the cells: () Blank PASN nanocapsules, (j) free docetaxel and (N) docetaxel-loaded PASN nanocapsules (n = 4).  p < 0.05: docetaxel-loaded PASN nanocapsules vs. free docetaxel.

Fig. 5. In vitro drug release profiles obtained from docetaxel-loaded PASN-CTAB (j) and PASN-BKC nanocapsules () in PBS, pH = 7.4 (mean ± S.D.; n = 3).

for up to 6 months. More specifically, the nanocapsules maintained their original particle size and zeta potential all throughout the study. This prolonged stability is in agreement with the stabilization role of the surfactant molecules entangled with the PASN molecules, altogether forming a protective shell around the oily nanodroplets. 3.3. Freeze-drying of nanocapsules Although PASN nanocapsules were stable in suspension for a few weeks, we explored the possibility of freeze-drying them in order to assess a long-term stability profile. For the setup of the experimental conditions, a previous freeze-drying feasibility study of chitosan nanocapsules was taken into account [15]. Thus, different concentrations of nanocapsules were selected for freeze-drying using different concentrations of the cryoprotectant agent trehalose (Fig. 4). The results indicated that the use of 5% trehalose made it feasible to freeze-dry the nanocapsules and reconstitute them without altering their particle size. 3.4. Encapsulation efficiency and release of docetaxel from PASN nanocapsules Docetaxel was selected as a model drug, to be encapsulated into the nanocapsules, due its hydrophobic character and its high efficacy in the treatment for a wide range of solid tumors [23]. As

Fig. 7. Effect on H460 cell viability of PASN nanocapsules prepared with the surfactant BKC (4 mg) after 2 h (A) or 48 h (B) of contact with the cells: () blank PASN nanocapsules, (j) free docetaxel and (N) docetaxel-loaded PASN nanocapsules (n = 4). # p < 0.005: docetaxel-loaded PASN nanocapsules vs. free docetaxel.


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Table 3 IC50 values of docetaxel in solution and included in PASN nanocapsules, obtained after incubation with H460 cells for 2 or 48 h. Data are the mean values ± SD of 4 independent experiments. Formulation

Free docetaxel Docetaxel-loaded PASN-CTAB nanocapsules Docetaxel-loaded PASN-BKC nanocapsules *

IC50 values (nM) 2 h Exposure

48 h Exposure

105.0 ± 9.0 30.9 ± 6.4* 26.2 ± 9.3*

36.40 ± 4.0 10.51 ± 1.3* 13.25 ± 0.6*

P < 0.001: docetaxel-loaded PASN NCs vs. free docetaxel.

Fig. 8. Uptake studies of fluorescent PASN nanocapsules in NCI-H460 cells assessed by FACS. Mean fluorescence intensity of DiD-loaded PASN-CTAB nanocapsules (j), blank PASN nanocapsules () DiD-loaded nanoemulsion (N) and fluorescent probe (DiD) dispersion ().  p < 0.05, DiD-loaded PASN nanocapsules vs. DiD dispersion and DiD-loaded nanoemulsion.

3.5. In vitro cytotoxicity of docetaxel-loaded PASN nanocapsules Cytotoxic studies were performed in order to assess the potential benefit of the nanocapsules for enhancing the activity of docetaxel. For this purpose, NCI-H460, a non-small lung cancer cell line, was used. The formulations tested were prepared with either CTAB or BKC surfactants. Figs. 6 and 7 show the viability profiles of NCIH460 cells after the exposure, for 2 and 48 h, to docetaxel-loaded PASN nanocapsules prepared with CTAB and BKC, respectively. The results showed that docetaxel cytotoxicity was increased almost 3 times, when presented in the nanoencapsulated form (p < 0.01). This increment in docetaxel cytotoxicity is also illustrated in the IC50 values summarized in Table 3. Based on IC50 values presented in Table 3, we could to infer that somehow, docetaxel was delivered into the cell in a more efficient way when encapsulated into the nanocapsules. As in the case of nanocapsules made of PEG [26], chitosan [16], or polyarginine nanocapsules [17], we could hypothesize that PASN are internalized into the cell and deliver their content at the intracellular level. This internalization might also be related to the asparagine intake need of lung cancer cells [10]. This targeting ability would, however, require further evaluation. On the other hand, the results presented in Fig. 6 also indicate that blank nanocapsules containing CTAB have a negligible toxicity whereas those containing BKC (Fig. 7) exhibited a dose-dependent toxicity profile upon incubation for up to 48 h. This difference in cytotoxicity could be attributed to the higher toxicity reported for BKC over CTAB [17], but also to the higher amounts of BKC that were required for the formation of stable nanocapsules (0.02% of CTAB vs. 0.04% for BKC). 3.6. Interaction of nanoparticles with cells

shown in Table 2, docetaxel could be efficiently encapsulated into the nanocapsules, reaching encapsulation values of around 75%, regardless of surfactant used. These results, which show the high capacity of PASN nanocapsules for the encapsulation of lipophilic drugs, are in agreement with those previously reported for other types of nanocapsules [16,17,24]. Following the encapsulation of docetaxel, an additional experiment was conducted in order to evaluate the release behavior of the nanocapsules upon incubation in phosphate buffer (pH = 7.4) in sink conditions. The release profile was characterized by an initial burst (releasing 55% of the drug) followed by a second phase of no release (Fig. 5). This two-phase release behavior has been typically observed for nanocapsules [16,25]. The initial burst of release could be attributed to the tendency of the system to reach the equilibrium between the oily and the aqueous phases, a tendency that was interrupted by the polymer coating.

In order to assess the potential of PASN nanocapsules for intracellular drug delivery, the interaction of fluorescently labeled (DiD) nanocapsules with NCI-H460 cells was evaluated using confocal fluorescent microscopy and flow cytometry. A DiD-loaded nanoemulsion (the same composition as nanocapsules but omitting the cationic surfactant and the polymer shell) and a DiD ethanolaqueous dispersion were used as controls. Prior to this study, a control experiment was made in order to assure that the loading of the fluorescent marker did not modify the physicochemical properties of the nanocapsules. The results of the FACS analysis are illustrated in Fig. 8. These results show that the PASN shell has a critical role in the association of PASN nanocapsules with NCI-H460 cells. Indeed, the fluorescence intensity upon cell treatment with PASN nanocapsules was 6 times higher than that observed upon incubation with the nanoemulsion or the fluorescent probe dispersion. On the other

Fig. 9. Confocal microscopic images of NCI-H460 cells after different treatments, (a) NCI-H460 cells without treatment (b) NCI-H460 after incubation for 2 h with DiD-loaded nanoemulsion and (c) Did-loaded PASN-CTAB nanocapsules. All images are projection of x–y sections, at a magnification of 63. Green channel: DiD signal of fluorescent treatments. Red channel: actin filaments counterstained with BodipyÒ phalloidin. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

G.R. Rivera-Rodríguez et al. / European Journal of Pharmaceutics and Biopharmaceutics 85 (2013) 481–487

hand, Fig. 9 shows the confocal microscopy images obtained upon 2 h incubation of the cells with the nanocapsules and the control formulations. The photographs evidence the green channel associated fluorescence spots in the case of cells treated with nanocapsules, whereas only a uniform red fluorescence is shown when the cells were incubated with the controls. These results provide further evidence of the interaction of PASN nanocapsules with the cells and also indicate that the polymer shell is critical role in this process. A similar behavior has already been described for other types of nanocapsules such as those made of chitosan [16] or polyarginine [27]; however, a specific advantage of PASN nanocapsules with regard to their use for intravenous administration relies on the negative charge associated with their PASN shell. Whether this interaction is driven by the asparagine avidity of cancer cells or not remains to be investigated. 4. Conclusions This report discloses a novel drug nanocarrier, PASN nanocapsules, consisting of an oily core and a PASN shell. The design of this nanocarrier was inspired by the known avidity of cancer cells by the asparagine amino acid. As expected, this nanocarrier exhibited attractive properties for the encapsulation and delivery of anticancer drugs, i.e. docetaxel. In addition, PASN nanocapsules were stable during long-term storage and also upon freeze-drying. Overall, these properties render this novel nanosystem a promising approach for the development of new nano-oncologicals. Acknowledgements The work has been supported by the Xunta de Galicia (PGIDIT 08CSA045209PR and Competitive Reference Groups-FEDER funds-Ref. 2010/18) and the European Commission (FP7 EraNet – EuroNanoMed Program-Instituto de Salud Carlos III; Lymphotarg proyect Ref. PS09/02670); G.R.R.R was in receipt of a CONACYTMexico scholarship. Authors also express their gratitude to Dr.Anxo Vidal for his help in FACS measurements. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejpb.2013.08.001. References [1] M.J. Alonso, P. Couvreur, Historical view of the design and development of nanocarriers for overcoming biological barriers, in: M.J. Alonso, N. Csaba (Eds.), Nanostructured Biomaterials for Overcoming Biological Barriers, The Royal Society of Chemistry, Cambridge, UK, 2012, pp. 3–36. [2] J.D. Byrne, T. Betancourt, L. Brannon-Peppas, Active targeting schemes for nanoparticle systems in cancer therapeutics, Advanced Drug Delivery Reviews 60 (2008) 1615–1626. [3] B. Mishra, B.B. Patel, S. Tiwari, Colloidal nanocarriers: a review on formulation technology, types and applications toward targeted drug delivery, Nanomedicine: Nanotechnology, Biology and Medicine 6 (2010) 9–24. [4] S. Parveen, R. Misra, S.K. Sahoo, Nanoparticles: a boon to drug delivery, therapeutics, diagnostics and imaging, Nanomedicine: Nanotechnology, Biology and Medicine 8 (2012) 147–166.


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