D d E A L a b c a A R R A A K S F P E P 1 m c t t c t r e i t t e v h 0 Colloids and Surfaces B: Biointerfaces 147 (2016) 442–449 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces jo ur nal ho me p ag e: www.elsev ier .com/ locate /co lsur fb evelopment of stained polymeric nanocapsules loaded with model rugs: Use of a fluorescent poly(phenyleneethynylene) stefânia V.R. Camposa,b, Jhones L. Oliveiraa, Sara A. Zavala-Betancourtc, ntonio S. Ledezmac, Eduardo Ariasc, Ivana Moggioc, Jorge Romeroc, eonardo F. Fracetoa,b,∗ Department of Environmental Engineering, State University of São Paulo, (UNESP), Sorocaba, SP, Brazil Department of Biochemistry, Institute of Biology, State University of Campinas (UNICAMP), Cidade Universitária Zeferino Vaz, Campinas, SP, Brazil Centro de Investigácion en Quimica Aplicada (CIQA), Blvd Enrique Reyna 140, 25294, Saltillo, Coahuila, Mexico r t i c l e i n f o rticle history: eceived 3 May 2016 eceived in revised form 26 July 2016 ccepted 19 August 2016 vailable online 21 August 2016 eywords: a b s t r a c t A phenyleneethynylene polymer (here denoted pPy3E-sqS) was synthesized and characterized by UV–vis spectroscopy, fluorescence spectroscopy, and TEM, and was used for the staining of polymeric nanocap- sules. The nanocapsules presented good temporal stability, without changes in shape or fluorescence, and were suitable for use in drug release systems. The mean particle size was around 430 nm, the polydis- persity index was below 0.2, and the zeta potential was around −13 mV. The release kinetics is one of the most important factors to consider in drug delivery systems, and here it was observed that nanocapsules tained nanocapsules luorescent polymer olymeric nanocapsules nvironmental applications henyleneethynylene polymer containing the fluorescent polymer still maintained the ability to modulate the release of the fungicides tebuconazole and carbendazim (used as model drugs) after 4 days. Preliminary results indicated that staining with the fluorescent pPy3E-sqS polymer could be used as a valuable tool to track the behav- ior of polymeric systems in the environment. However, further studies will be needed to clarify the environmental behavior and possible toxicity. © 2016 Elsevier B.V. All rights reserved. . Introduction There is increasing interest in the study and application of nano- aterials in agriculture, as one of the approaches to resolving global hallenges such as population growth, climate change, and the need o improve productivity while at the same time decreasing harm to he environment [1,2]. The application of nanotechnology in agri- ulture aims to reduce applications of agrochemicals, detect and reat diseases, improve the uptake of nutrients by plants, direct the elease of active ingredients towards specific sites, and mitigate ffects on non-target plants [2–6]. Due to the increased use of nanomaterials in various fields, ncluding agriculture, there is a need for thorough knowledge of he interactions of these substances with both target and non- arget plants, as well as to follow their behavior in the environment, specially considering their toxicity [7–10]. To this end, the use ∗ Corresponding author at: Department of Environmental Engineering, State Uni- ersity of São Paulo, (UNESP), Sorocaba, SP, Brazil. E-mail address: leonardo@sorocaba.unesp.br (L.F. Fraceto). ttp://dx.doi.org/10.1016/j.colsurfb.2016.08.031 927-7765/© 2016 Elsevier B.V. All rights reserved. of stained nanocapsules has shown promise for investigating the interactions of nanomaterials with various matrices. The use of nanocapsule systems stained with fluorescence dyes is a common approach employed to study cells and tissues in vivo and in vitro [11–13]. The advantages of using fluorescence tech- niques include high sensitivity, rich contrast, easy staining, and the use of standard analytical methods [14–16]. Various dyes used to stain nanomaterials exhibit high quantum yields, but in many cases, problems of cytotoxicity have limited their broad application in biological studies [14–16]. It is therefore important to develop and evaluate new materials with potential for use in future applications in areas including agriculture, envi- ronment, and bio-medicine. The chain structures of conjugated polymers show alternation of single and double or triple bonds, creating extended delocal- ization of electrons and consequently the generation of electronic properties [17–19]. The excitation energies of these �-conjugated materials are usually in the visible range, making them optically active [20]. In this work, we report the preparation and characterization of polymeric nanocapsules consisting of polycaprolactone and dx.doi.org/10.1016/j.colsurfb.2016.08.031 http://www.sciencedirect.com/science/journal/09277765 http://www.elsevier.com/locate/colsurfb http://crossmark.crossref.org/dialog/?doi=10.1016/j.colsurfb.2016.08.031&domain=pdf mailto:leonardo@sorocaba.unesp.br dx.doi.org/10.1016/j.colsurfb.2016.08.031 E.V.R. Campos et al. / Colloids and Surfaces B: Biointerfaces 147 (2016) 442–449 443 N N O S OC12H25 C12H25O S S O n N Br O OH + HS S SH2(X) Br N O S S S O N Br OC12H25 C12H25O H H+ 1 2 3 4 5 a b 68% 72% ◦C, 15 s w a o a o w w f c fl T p a t s s e C 2 2 A c i b d d m t a 2 c c 3 2 S c 1 C f Scheme 1. Reagents and conditions: (a) DCC/DMAP, CH2Cl2, 0 to r.t. tained with a fluorescent poly(phenyleneethynylene) sequenced ith flexible thioester-diethyldisulfide chains. This system, here- fter denoted pPy3E-sqS, has the potential to enable the detection f nanocapsules by UV/Vis and fluorescent spectroscopy, as well s by laser scanning confocal microscopy (LSCM). The feasibility f different formulations of nanocapsules, unstained or stained ith pPy3E-sqS, were evaluated as carrier systems by loading them ith tebuconazole and carbendazim. These compounds, which are ungicides, were used here as model drugs. The physicochemi- al stabilities and the release profiles of the loaded and unloaded uorescent nanocapsules were assessed as a function of time. his study opens perspectives for monitoring the interactions of olymeric nanocapsules with target and non-target organisms in griculture, such as their penetration into leaves, interaction with he soil matrix, and diffusion to water resources, in order to under- tand the ways in which nanocapsules interact with these complex ystems. In addition, nanocapsules stained with pPy3E-sqS could be mployed to evaluate the toxicity in soil (such as towards worms – aenorhabditis elegans), plants, and insects. . Materials and methods .1. Materials The following chemical products were obtained from ldrich and were used without further purification: poly- aprolactone (PCL), poly(vinyl alcohol) (PVA), copper(I) odide, dichlorobis(triphenylphosphine) palladium(II), 6- romopyridine-3-carboxylic acid, 2-mercaptoethylsulfide, sodium iethyldithiocarbamate, dicyclohexylcarbodiimide (DCC), and imethylaminopyridine (DMAP). CH2Cl2, CHCl3, hexane, and ethanol were purchased from Aldrich and JT Baker. THF and riethylamine were distilled from sodium/benzophenone complex nd KOH, respectively. .2. Synthesis of pPy3E-sqS S,S’-2,2’-thiobis(ethane-2,1-diyl) bis(6-bromopyridine-3- arbothioate) (compound 3 in Scheme 1) A round bottom flask fitted with a stirrer, condenser, and alcium chloride guard tube was loaded with 6-bromopyridine- -carboxylic acid (3.3 g, 16.33 mmol) (compound 1 in Scheme 1), -mercaptoethylsulfide (0.96 mL, 7.42 mmol) (compound 2 in cheme 1), and 80 mL of CH2Cl2 (methanol free). The solution was ooled to ∼0 ◦C, followed by dropwise addition of DCC (3.306 g, 6.0 mmol) and DMAP (55 mg, 0.445 mmol) dissolved in 10 mL of H2Cl2. The mixture was then heated at 50 ◦C overnight. The urea ormed was filtered off and the filtrate was washed with a 0.5 M HCl h; (b) [(C6H5)3P]2PdCl2 (2.5 mol%), CuI (1.5 mol%), Et3N, 80 ◦C, 36 h. solution. The mixture was transferred to a separating funnel and the organic layer was extracted with CH2Cl2 and dried with MgSO4. After filtering off the drying agent, the solvent was removed under vacuum and the crude product was chromatographed through a silica gel column using CH2Cl2 as eluent, resulting in a 68% yield of a white powder with the following characteristics: mp 141–143 ◦C; 1H NMR (CDCl3, 300 MHz) � (ppm): 8.8 (s, 2H, H-Ar), 8.0 (d, 2H, H- Ar), 7.62 (d, 2H, H-Ar), 3.35 (t, 4H, −COS-CH2), 2.9 (t, 4H, −CH2-S-); 13C NMR (CDCl3, 75 MHz) � (ppm): 189.06 (C O), 148.93 (N-C2), 147.23 (C6-Br), 136.61 (C4), 131.57 (C3), 128.30 (C5), 31.59 (-CO- S-CH2-), 29.29 (-CH2-S-). Degassed Et3N (40 mL) was added through a dry cannula, under a nitrogen atmosphere, to a two-neck round bottom flask containing compound 3 (220 mg, 0.42 mmol), [(C6H5)3P]2PdCl2 (7.8 mg, 0.11 mmol), and CuI (ca. 1.0 mg). The mixture was heated at 60 ◦C for 15 min. Subsequently, 1,4-diethynyl-2,5- bis(dodecanoxy)benzene (compound 4) (189 mg, 0.38 mmol) dissolved in dry and degassed Et3N (5 mL) was added using a syringe, followed by stirring for 36 h at 80 ◦C. After cooling, the mixture was filtered to eliminate the ammonium salt, washed with THF, and the organic phase was evaporated to a minimal volume prior to precipitation with methanol (50 mL) containing sodium diethyldithiocarbamate21 (15 mg), followed by centrifugation. The pasty residue was recovered into 4 mL of CHCl3 and precipitated twice in clean methanol. Finally, the product was passed through a preparative SEC column (Bio-Beads SX1, Bio-Rad) using toluene as eluent. It was then concentrated, dissolved in 10.5 mL of CHCl3, and refrigerated until used. The yield was 72%, �abs (�) = 408 nm (79.98 g L−1 cm−1), and �fluo (�) = 480 nm (0.30 ± 0.02). 2.3. Characterization of pPy3E-sqS A JEOL Eclipse spectrometer was used to acquire 1H NMR (300 MHz) and 13C NMR (75.4 MHz) spectra at room temperature, using CDCl3 as the solvent and internal standard. The molecular weight of the polymer was determined by GPC, using an Agilent Technologies 1200 series HPLC (Model 61316A) with PS stan- dards, refractive index detector, and toluene as the mobile phase at 45 ◦C and 1 mL min−1. For the photophysical characterization of the polymer, spectroscopic grade chloroform (Sigma-Aldrich) was used. UV–vis spectra were acquired with an Agilent 8453 spec- trophotometer. Fluorescence emission and excitation spectra were obtained using a Perkin Elmer LS50 B spectrofluorimeter operated at an excitation wavelength of 394 nm. The temperature was con- trolled at 25.0 ± 0.3 ◦C by means of a recirculating water bath. The fluorescence quantum yield was determined according to the literature, using 0.1 M H2SO4 quinine sulfate (� = 0.54)[22] as a standard. Three different samples with optical density at 394 nm 4 faces l L ( A T o � w � f 2 p o a i w e 1 8 r c m d 1 i t p 2 o p e u fi a a f e w w 9 s P A r n r o t w A a a 44 E.V.R. Campos et al. / Colloids and Sur ower than 0.1 were analyzed, and the values were averaged. ifetimes were determined with a flash photolysis spectrometer LP920 KS, Edinburgh Instruments). A 0.01% suspension of Ludox S40 (Aldrich) in ultrapure water was used for the prompt signal. he data were fitted using the instrument software. The average lifetime (�) was calculated from the lifetimes btained (�i), using the following equation: = ˙n 1 fi�i ,(1) here fi is the fractional fluorescence contribution of each lifetime i, given by: i = ˛i�i ˙n 1˛i�i ,(2) and � is the pre-exponential coefficient obtained from the fit. .4. Preparation of the polymeric nanocapsules The emulsion/solvent evaporation technique was employed to repare the polymeric nanocapsules [24]. Briefly, a known amount f the pPy3E-sqS fluorescent polymer was dissolved in 10 mL of cetone. Simultaneously, another solution was prepared contain- ng 400 mg of PCL dissolved in 20 mL of chloroform. These solutions ere emulsified by ultrasonication for 1 min at 90 W. This primary mulsion was added to an aqueous solution (50 mL) containing 50 mg of PVA, and the resultant solution was ultrasonicated for min to form a double emulsion. Finally, the organic solvent was emoved with the aid of a rotary evaporator and the emulsion was oncentrated to a final volume of 10 mL. Taking into account the ass of PCL polymer used in the preparation of the nanocapsules, ifferent concentrations of the fluorescent pPy3E-sqS polymer (0.5, , 2, 3, and 4% w/w) were added to the formulations in order to dentify the concentration that resulted in the greatest coverage of he nanocapsules. Nanocapsules without fluorescent polymer were repared as a control. .5. Polymeric nanocapsules characterization Characterization of the nanocapsules was performed using flu- rescence spectra obtained for the samples immediately after reparation and following storage under refrigeration for differ- nt time intervals. Zeta potential measurements were carried out sing a ZetaSizer Nano ZS90 analyzer (Malvern Instruments), at a xed angle of 90◦ and temperature of 25 ◦C. NTA analyses employed laser with a wavelength of 532 nm (green), a CMOS camera, nd NanoSight v. 2.3 software [25]. Electron microscopy was per- ormed using an FEI Titan 300 kV field emission gun microscope quipped with an S-TWIN symmetrical condenser-objective lens ith a spherical aberration coefficient (Cs) of 1.25 mm. The images ere acquired with a CCD camera. A Zeiss-LEO microscope (Model 06) was employed to investigate the morphology of the nanocap- ules. Laser scanning microscopy images were acquired with a Zeiss ascal 5 instrument, with excitation using an argon 458 nm laser. two-channel configuration was chosen in order to obtain fluo- escence and reflection images. In the fluorescence image, only the anocapsules coated with the polymer could be seen, while the eflection image showed the entire sample. The color of the flu- rescence was selected from the palette of the software and was herefore not a direct visualization of the emission. The samples ere prepared using polymer concentrations of 0.5, 1, 2, 3, and 4%. 10 �L aliquot of each suspension was diluted in 10 mL of water, nd 0.1 mL of this preparation was dropped onto a microscopic slide nd left to dry for one day at room temperature, prior to the analysis. B: Biointerfaces 147 (2016) 442–449 2.6. Release experiments Drug release kinetic assays were performed in order to deter- mine whether addition of the fluorescent pPy3E-sqS influenced the profile of release from the nanocapsules [26]. For this pur- pose, carbendazim and tebuconazole were used as model drugs, in order to enable comparison with previously published results [27]. The release assays were performed under dilution sink conditions, with constant stirring. Appropriate amounts of the formulations were diluted in the central acceptor compartment (using deionized water). Aliquots were collected and transferred to Falcon tubes at intervals of 15, 30, and 60 min during the first eight hours, after which two aliquots were obtained every 24 h, up to 96 h. The sam- ples were centrifuged in order to sediment out the nanocapsules, and the concentrations of the compounds were determined by HPLC. 3. Results and discussion 3.1. Polymer synthesis and characterization The semi-rigid polymer (compound 5), here denoted pPy3E- sqS, was synthesized according to the chemical route given in Scheme 1. The pathway essentially consisted of two reactions: i) thioesterification of 6-bromopyridine-3-carboxylic acid (1) with 2- mercaptoethylsulfide (2) under the N,N’-dicyclohexylcarbodiimide (DCC)/DMAP-mediated esterification conditions, in CH2Cl2, to give S,S’-2,2’-thiobis(ethane-2,1-diyl) bis(6-bromopyridine-3- carbothioate) (3), with a yield of 68%; ii) the Pd/CuI cross-coupling reaction between the corresponding dibromide terminated monomer (3) and 1,4-diethynyl-2,5-bis(dodecanoxy)benzene (8) [15]. The use of a co-solvent was avoided, because it would drastically increase the molecular weight, which usually gives rise to insoluble aggregates. The polymer was submitted to a metal decomplexation process by precipitation, once with a methanol/sodium dithiocarbamate solution, and twice with clean methanol. The shortest oligomers were eliminated using a preparative SEC column (Bio-Beads SX1, Biorad), with toluene as eluent. The proton resonance peaks of pPy3E-sqS were broader, compared to those of the corresponding monomers; the acetylenic proton disappeared, while the protons near the bromine could be identified, suggesting that the molec- ular chains were mainly terminated by bromines (Fig. 1S). The polymer (5) was soluble in chloroform, THF, and toluene. It was partially soluble in dipolar solvents such as dimethylformamide and dimethylsulfoxide, and was precipitated in polar solvents such as methanol and ethanol. The average molecular weight, calculated using PS standards, was 26504 g/mol, and the polydispersity index was 2.99 (typical of polycondensed conjugated polymers). The polymer presented conjugated and fluorescent phenyleneethyny- lene units alternating with flexible sequences containing sulfur atoms, imparting a partially amphiphilic character that improved its solubility in common organic solvents, preventing chain aggre- gation due to �-� electronic interactions and allowing interaction with poly(caprolactone). Fig. 1A shows the UV–vis, excitation, and fluorescence spectra in CHCl3. The polymer exhibited a main absorption peak at 403 nm, which could be attributed to HOMO-LUMO electronic transitions, and a higher energy peak at 324 nm, probably due to the HOMO- 1/LUMO transition associated with the alkoxy substitution in the phenyleneethynylene. The emission spectrum presented a broad band with maximum at 480 nm, at any excitation wavelength, as shown in Fig. 1B. The excitation spectrum corresponded to the absorption spectrum, as can be seen in Fig. 1A. These two latter fea- tures indicated that the emission was always generated by the same E.V.R. Campos et al. / Colloids and Surfaces B: Biointerfaces 147 (2016) 442–449 445 F ra of t c ces to e c ( p a p v o D f r c w b i t t a s 3 p fi t s ig. 1. UV–vis (black line), excitation (red line), and fluorescence (blue line) spect hloroform, at different excitation wavelengths (B). (For interpretation of the referen lectronic transition and that the two absorption bands were asso- iated with only one chromophore. The fluorescence quantum yield � = 0.30) was on the same order of magnitude reported for other oly(phenyleneethynylene)s, and somewhat lower than found for PPE with similar sequence. The fluorescence decay showed a bi-exponential fit (Fig. S2, Sup- orting information), with an average lifetime of 1.69 ns. From the alues of quantum yield and lifetime, a non-radiative constant (knr) f 0.41 ns and a radiative constant (krad) of 0.18 ns were obtained. espite the high absorption coefficient (� = 79.98 g L−1 cm−1), the act that knr was greater than krad was in agreement with the fluo- escence quantum yield and revealed that de-excitation processes ompeted with fluorescence. As the Stokes’s shift (�n = 4014 cm−1) as quite large, internal conversion was probably one of the possi- le de-excitation mechanisms. However, the possible existence of ntersystem crossing could not be excluded, especially as bromine erminal groups were present (as indicated by the NMR spec- ra). Overall, a quantum yield of 30%, together with the partially mphiphilic character of the polymer, indicated that pPy3E-sqS hould be a good candidate for use as a fluorescent dye. .2. Staining and characterization of the nanocapsules Different weight percentages of pPy3E-sqS were added to the oly(epsilon-caprolactone) nanocapsules and the samples were rst analyzed by LSCM microscopy. As an example, Fig. 2 shows he fluorescence and reflection images obtained for nanocapsules tained with 3% of the fluorescent pPy3E-sqS. The images for the Fig. 2. LSCM fluorescence (left) and reflection (right) images of n he pPy3E-sqS polymer in chloroform (A); Fluorescence spectra of the polymer in colour in this figure legend, the reader is referred to the web version of this article.) other polymer percentages are provided in the Supporting infor- mation (Fig. S3). The poly(epsilon-caprolactone) nanocapsules did not emit under the excitation conditions used, as can be observed for the image corresponding to 0% of polymer. The nanocapsules appeared to be quasi–spherical, although some particles coalesced due to the drying process that was necessary for the LSCM analyses. At 0.5%, practically no emission could be seen, while fluorescent nanocapsules began to appear at a polymer concentration of 1%. The quantity of fluorescent spots increased with the polymer concen- tration, and at 3% all of the nanocapsules present in the reflection image could also be seen in the fluorescence channel. At 4%, there was a clear decrease in fluorescence intensity, indicative of reab- sorption effects. Fluorescence spectra of the different suspensions were acquired separately with a fluorimeter. Based on the LSCM observations, the spectrum for the 0.5% suspension was not included in Fig. 3, which illustrates the fluo- rescence spectra of the particles with 1, 2, 3, and 4% of polymer dissolved in distilled water. These spectra showed a red shift, relative to the spectrum of the polymer in chloroform (Fig. 1B), which could be explained by the solid-state effects reported for conjugated polymers, in particular if the polymer is considered to interact with the capsule as a thin layer. There was also a red shift as the percentage of polymer increased, which could again be attributed to solid-state effects, with increased thickness of the polymer layer coating resulting in decreased intensity. Despite the fact that UV–vis spectra could not be recorded, due to the turbid- ity of the suspensions, the red shift suggested that the thickness increased, so an increase in the absorption was expected. Hence, anocapsules coated with 3% of the fluorescent pPy3E-sqS. 446 E.V.R. Campos et al. / Colloids and Surfaces 640600560520480440 0 20 40 60 80 100 120 In te ns ity (a .u .) Wavelength (nm) 1 % 2 % 3 % 4 % F c t I u p o a t p a p a r t a i g p o p n n r o a r i n n t I o t o I m s b 3 f i i ig. 3. Fluorescence spectra of the nanocapsules stained using different polymer oncentrations. he fluorescence quenching was probably due to self-absorption. n terms of fluorescence intensity, the best results were obtained sing 1% polymer. However, the LSCM results indicated that com- lete coating of the particles was only achieved with concentrations f 3% and above. As the intensity was dramatically quenched at 4%, concentration of 3% was taken as the optimum, considering both he spectroscopic and microscopic characterizations. In order to obtain further information concerning the staining rocess, the pPy3E-sqS polymer and the stained nanocapsules were nalyzed using HRTEM. Fig. 4a shows a planar view of a film of Py3E-sqS with a calculated periodicity of 0.25 ± 0.02 nm. Selected rea electron diffraction (SAED) analysis (Fig. 4a insert) showed a eflection at d = 0.25 nm. These distances correspond to the separa- ion between conjugated chains, where the dodecanoxy side chains re lengthened perpendicularly to the film surface, as indicated n Fig. 4b, suggesting that strong �-� interaction between conju- ated segments predominated in this polymer, as usually found for henyleneethynylene macromolecules [28]. In order to investigate the effect of staining with the polymer n the nanocapsules, and to determine whether 3% of fluorescent olymer was sufficient to provide full coverage, a drop of stained anocapsules was observed by TEM (Fig. 4c), and a section of one anocapsule was submitted to HRTEM analysis (Fig. 4d). Three egions could be clearly identified: i) a region with periodicity f 0.25 nm, similar to that of the conjugated polymer backbones ssembled parallel to the surface, as previously discussed; ii) a egion where the polymer backbones lay flat on the surface; and ii) amorphous regions associated with the poly(caprolactone) anocapsule wall (shown with an arrow), indicating that the anocapsules were not totally covered by the pPy3E-sqS. The illus- ration in Fig. 4e provides a visual overview of the staining process. ncreasing the content of polymer led to a tendency of the polymer n the nanocapsule surface to self-assemble, hence increasing the hickness, which was consistent with the greater quenching of flu- rescence at higher polymer concentrations, described previously. n other words, the self-assembling of the polymer to form blocks of olecules was stronger than its interaction with the nanocapsule urface. This hypothesis will be confirmed using a similar polymer, ut with glycol side chains (currently under synthesis). .3. Colloidal stability of the stained polymeric nanocapsules Before investigation of the release profiles of the test compounds rom the nanocapsules coated with pPy3E-sqS, the particle stabil- ty was evaluated by measuring the mean diameter, polydispersity ndex, zeta potential, and fluorescence intensity, as a function of B: Biointerfaces 147 (2016) 442–449 time (up to 90 days). The mean diameter obtained by DLS for the nanocapsules without pPy3E-sqS (Fig. 5a, black columns) was 249.3 ± 3.4 nm immediately after preparation and remained with- out significant changes during the storage period, with a value of 245.6 ± 5.1 nm after 90 days. The polydispersity index (Fig. 5a, black line) of the nanocapsules remained below 0.2 throughout the storage period, indicating good homogeneity of the nanocap- sule size distribution. The nanocapsules with pPy3E-sqS (Fig. 5a, red column) showed a greater mean diameter (428.5 ± 4.1 nm), compared to the control nanocapsules, and maintained this size distribution range during the 90 days of storage, without any sig- nificant changes. This increase in the mean diameter was indicative of incorporation of the fluorescent polymer in the polymeric net- work of the nanocapsules, as previously discussed for the HRTEM images. The polydispersity index (Fig. 5a, red line) showed a value of 0.29 ± 0.05 immediately after preparation, then decreased to 0.2 after 15 days and subsequently remained constant up to 90 days of storage Measurement of the surface charge of nanocapsules, known as the zeta potential, provides an indication of the stability of colloidal systems; higher values (positive or negative) are indica- tive of greater stability. The initial zeta potentials (Fig. 5b) were −13.1 ± 1.3 mV and −6.9 ± 1 mV for nanocapsules with and with- out the fluorescent polymer, respectively, and the values increased after 90 days of storage. However, the surfactant used during the synthesis of the nanocapsules was poly(vinyl alcohol), which is known to stabilize colloidal systems by steric hindrance, so elec- trostatic stabilization was not the main mechanism responsible for stabilization of the formulations [21,22,23,29]. The mean diameters of the nanocapsules with and without pPy3E-sqS were measured using nanoparticle tracking analysis (NTA), as a function of storage time. Unlike the PSC technique, which determines the size distribution of a set of nanoparticles, NTA analyzes each particle individually. In addition to the size distribution, this technique is able to determine the concentra- tion of nanocapsules present in the formulation (Fig. 6). For the nanocapsules without pPy3E-sqS, the same mean diameters were obtained by both techniques (DLS and NTA). The concentration of the control nanocapsules was around (5.60 ± 0.65) x 1012 parti- cles/mL, and remained almost constant during 90 days. There was no formation of aggregates (which would have been observed if the concentration of the nanocapsules decreased over time). Unlike the control nanocapsules, in the case of the nanocapsules with the fluorescent polymer, a smaller diameter was obtained using NTA (286.6 ± 5.40 nm), compared to DLS (428.5 ± 4.11 nm). Although both techniques measure Brownian motion, there is a difference in the nature of the analysis. NTA identifies and tracks the movement of the particles on a particle-by-particle basis, while DLS does not observe the particle, but instead uses the dispersion in light inten- sity caused by constructive and destructive interferences, hence explaining the difference between the two techniques in terms of particle size. The nanocapsules with pPy3E-sqS showed a concen- tration of (7.05 ± 0.23) × 1012 particles/mL. Finally, the fluorescence of the pPy3E-sqS in the formulation was characterized using spectroscopic analysis. The results (Fig. 7) showed that there was a slight decrease in fluorescence intensity for the nanocapsules with 3% of pPy3E-sqS, with an approximately 20% reduction in intensity after 90 days of storage. 3.4. Release kinetics of model drugs Two model molecules (carbendazim and tebuconazole) were employed to evaluate whether the presence of pPy3E-sqS altered the release kinetics, compared to the values reported previously for nanocapsules without pPy3E-sqS [27]. E.V.R. Campos et al. / Colloids and Surfaces B: Biointerfaces 147 (2016) 442–449 447 Fig. 4. (a) HRTEM image of a film of pPy3E-sqS polymer deposited on a Lacy carbon grid, with (insert) the SAED pattern of the corresponding film. Both images show a periodicity of 0.25 nm; (b) Illustration showing the correspondence between the periodicity of 0.25 nm and the distance between planes of conjugated segments; (c) TEM image of a nanocapsule with the fluorescent pPy3E-sqS polymer; (d) HRTEM image of one section of the nanocapsule; the arrow indicates the amorphous morphology of an uncoated nanocapsule section, while the magnified image shows the same periodicity of 0.25 nm found for the pPy3E-sqS polymer; (e) Sketch of the nanocapsule, showing self-assembly of the pPy3E-sqS polymer in two configurations. Fig. 5. (A) Stability of hydrodynamic diameter for nanocapsules without fluorescent pPy3E-sqS (red column) and nanocapsules with pPy3E-sqS (black column), and poly- dispersity index for nanocapsules without fluorescent pPy3E-sqS (red line) and nanocapsules with pPy3E-sqS (black line). (B) Zeta potential for the nanocapsules without pPy3E-sqS (red column) and nanocapsules with pPy3E-sqS (black column). The values represent the means of three experiments (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 6. Mean diameter (A) and concentration (B) of the nanocapsules, measured using NTA, as a function of storage time (0, 15, 30, 60, and 90 days). The values represent the means of three experiments (n = 3). 448 E.V.R. Campos et al. / Colloids and Surfaces 420 470 52 0 57 0 62 0 67 0 72 0 0 20 40 60 80 0 15 30 45 60 75 90 40 50 60 70 80 0 d 15 d 30 d 60 d 90 d In te ns ity (a .u .) Wavelength (nm ) F lu or es ce nc e at 5 15 n m Days Fig. 7. Time-dependent fluorescence intensity of the nanocapsules stained with 3% of fluorescent pPy3E-sqS polymer. Insert: fluorescence intensity at 515 nm, plotted against time. F c t r 6 o w p f s a a t H r w c c n 4 o o d c c e t n w [ nanoparticles on human and environment: review of toxicity factors, ig. 8. Cumulative release of carbendazim and tebuconazole from nanocapsules ontaining the fluorescent pPy3E-sqS polymer. The values represent the means of hree experiments (n = 3). The results (Fig. 8) showed that for both compounds, there was a apid release from the interior of the nanocapsules during the first 00 min, followed by sustained release. After 5760 min, around 30% f the carbendazim incorporated in the nanocapsules was released, hile around 50% of the tebuconazole was released over the same eriod. A possible explanation for the MBC release profile is that rom 0 to 400 min, the MBC molecules that were associated more uperficially with the nanocapsules were released. Between 400 nd 4800 min, the cumulative release remained constant, while fter 4800 min, the MBC molecules that had been inserted into he nanocapsule cores began to be released to the environment. ence, there was slow release of the encapsulated MBC. These esults were in agreement with those obtained for nanocapsules ithout pPy3E-sqS, where 20% of carbendazim and 46% of tebu- onazole were released after 5760 min [27]. This indicated that the onjugated polymer did not affect the process of release from the anocapsules. . Conclusions and perspectives This work describes the synthesis and characterization of a flu- rescent conjugated polymer and its incorporation on the surface f polymeric nanocapsules, with the aim of developing an optical etection system for the tracking and monitoring of nanoparti- les in the environment. Considering that incorporation of the onjugated polymer might cause destabilization, the system was valuated using physicochemical characterization and investiga- ion of the kinetics of release of active agents from the stained anocapsules. A semi-rigid rod-like poly(phenyleneethynylene) sequenced ith flexible thioester-diethyldisulfide chains, denoted pPy3E-sqS, [ B: Biointerfaces 147 (2016) 442–449 was synthesized by the Sonogashira cross-coupling reaction. In chloroform, the pPy3E-sqS polymer emitted high energy light in the blue region at 480 nm, with a high quantum yield, making it a good candidate for staining nanocapsules. Analyses using LSCM and HRTEM microscopy confirmed that the fluorescent polymer (pPy3E-sqS) interacted with the poly- meric wall of the nanocapsules. The stained nanocapsules showed a mean diameter of around 428.5 ± 4.1 nm and remained stable over a period of 90 days. NTA and TEM analysis showed that the nanocapsules were spherical, with an average concentration of (7.05 ± 0.23) × 1012 particles/mL. The release profiles of the model drugs were not affected by the presence of the fluorescent polymer. The nanocapsules provided efficient drug release and remained stable during storage. Therefore, they could be used in many mon- itoring applications, including those involving the interaction of nanocapsules with target and non-target organisms. These systems may be used in different areas such as health, biotechnology, and agriculture, amongst others. Further work will be needed in order to characterize the interactions of the nanocapsules with model plants, as well as to evaluate their toxicity. Acknowledgment The authors are grateful for the support provided by the São Paulo Science Foundation, Brazil (FAPESP, grants #2014/20273- 4, #2014/20286-9, #2013-12322-2 and 2015/15617-9), Mexican National Council for Science and Technology (CONACYT, México) for the financial support through the project 229761/INFR-2014-02 that allowed the acquirement of the Flash photolysis equipment. Also the authors would like to thanks the bilateral cooperation project between Brazil-Mexico, join Cooperation CONACYT-CNPq (Project:174806) as well we gratefully acknowledge CONACYT’s support to the present work through project 270878, Laboratorio Nacional de Materiales Grafénicos. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.08. 031. References [1] S.S. Mukhopadhyay, Nanotechnology in agriculture: prospect and constraints, Nanotechnol. Sci. Appl. 7 (2014) 63–71. [2] M. Kah, Nanopesticides and nanofertilizers: emerging contaminants or opportunities for risk mitigation, Front. Chem. 3 (2015) 1–5. [3] B.P. Sekhon, Nanotechnology in agri-food production: an overview, Nanotechnol. Sci. Appl. 7 (2014) 31–53. [4] C. Parisi, M. Vigani, E. Rodriguez-Cerezo, Agricultural nanotechnologies: what are the current possibilites? Nanotoday 10 (2015) 124–127. [5] E.V.R. Campos, J.L. Oliveira, L.F. Fraceto, Applications of controlled release system for fungicides, herbicides, acaricides, nutrients, and plant growth, hormones: a review, Adv. Sci. Eng. Med. 6 (2014) 1–15. [6] E.V.R. Campos, J.L. Oliveira, L.F. Fraceto, B. Singh, Polysaccharides as safer release system for agrochemicals, Agron. Sustain. Dev. 35 (2015) 47–66. [7] S.J. Klaine, P.J.J. Alvarez, G.E. Batley, T.F. Fernades, R.D. Handy, D.Y. Lyon, S. Mahendra, M.J. McLaughlin, J.R. Lead, Nanomaterials in the environment: behavior, fate, bioavailability, and effects, Environ. Toxicol. Chem. 27 (2008) 1825–1851. [8] M.A. Maurer-Jones, I.L. Gunsolus, C.J. Murphy, C.L. Haynes, Toxicity of engineered nanoparticles in the environment, Anal. Chem 85 (2013) 3036–3049. [9] K.L. Garner, A.A. Keller, Emerging patterns for engineered nanomaterials in the environment: a review of fate and toxicity studies, J. Nanopart. Res. 16 (2014) 2503. 10] M. Sajid, M. Ilyas, C. Basheer, M. Tariq, M. Daud, N. Baig, F. Shehzad, Impact of exposures, control strategies, and future prospects, Environ. Sci. Pollut. Res. 22 (2015) 4122–4143. 11] L. Shang, K. Nienhaus, G.U. Nienhaus, Engineered nanoparticles interacting with cells: size matters, J. Nanobiotechnology. 12 (2014) 1–11. http://dx.doi.org/10.1016/j.colsurfb.2016.08.031 http://dx.doi.org/10.1016/j.colsurfb.2016.08.031 http://dx.doi.org/10.1016/j.colsurfb.2016.08.031 http://dx.doi.org/10.1016/j.colsurfb.2016.08.031 http://dx.doi.org/10.1016/j.colsurfb.2016.08.031 http://dx.doi.org/10.1016/j.colsurfb.2016.08.031 http://dx.doi.org/10.1016/j.colsurfb.2016.08.031 http://dx.doi.org/10.1016/j.colsurfb.2016.08.031 http://dx.doi.org/10.1016/j.colsurfb.2016.08.031 http://dx.doi.org/10.1016/j.colsurfb.2016.08.031 http://dx.doi.org/10.1016/j.colsurfb.2016.08.031 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0005 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0005 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0005 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0005 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0005 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0005 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0005 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0005 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0005 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0005 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0005 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0005 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0005 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0005 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0005 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0005 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0010 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0015 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0015 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0015 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0015 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0015 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0015 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0015 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0015 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0015 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0015 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0015 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0015 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0015 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0015 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0015 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0015 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0020 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0025 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0030 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0035 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0040 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0045 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0050 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0055 faces [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ E.V.R. Campos et al. / Colloids and Sur 12] C. Simonsson, G. Bastiat, M. Pitorre, A.S. Klymchenko, J. Béjaud, Y. Mély, J.-P. Benoit, Inter-nanocarrier and nanocarrier-to-cell transfer assays demonstrate the risk of an immediate unloading of dye from stained lipid nanocapsules, Eur. J. Pharm. Biopharm. 98 (2016) 47–56. 13] M.-H. Nguyen, J.-S. Lee, I.-C. Hwang, H.-J. Park, Evaluation of the penetration of nanocarriers into red pepper leaf using confocal laser scanning microscopy, Crop Prot. 66 (2014) 61–66. 14] J. Yan, M.C. Estévez, J.E. Smith, K. Wang, X. He, L. Wang, W. Tan, Dye-doped nanoparticles for bioanalysis, Nanotoday 3 (2007) 44–50. 15] A.B. Chinen, C.M. Guan, J.R. Ferrer, S.N. Barnaby, T.J. Merkel, C.A. Mirkin, Nanoparticles probes for the detection of cancer biomarkers, cells, and tissues by fluorescence, Chem. Rev. 115 (2015) 10530–10574. 16] O.S. Wolfbeis, An overview of nanoparticles commonly used in fluorescent bioimaging, Chem. Soc. Rev. 44 (2015) 4743–4768. 17] G. Castruita, E. Arias, I. Moggio, F. Pérez, D. Medellín, R. Torres, R. Ziolo, A. Olivas, E. Giorgetti, M. Muniz-Miranda, Synthesis, optical properties and supramolecular order of �-conjugated 2 5-di(alcoxy) phenyleneethynylene oligomers, J. Mol. Struct. 936 (2009) 177–186. 18] T. Maillou, J. Le Moigne, V. Dumarcher, L. Rocha, B. Geffroy, J.-M. Nunzi, Oligo (phenylethynylene) as a high photoluminescence quantum yield material and its distributed feedback laser emission in thin films, Adv. Mat. 14 (2002) 1297–1301. 19] R.H. Friend, R.W. Gymer, A.B. Holmes, J.H. Burroughes, R.N. Marks, C. Taliani, D.D.C. Bradley, D.A. dos Santos, J.L. Brédas, M. Lögdlund, W.R. Salaneck, Electroluminescence in conjugated polymer, Nature 397 (1999) 121–128. 20] P. Karam, A.A. Hariri, C.F. Calver, X. Zhao, K.S. Schanze, G. Cosa, Interaction of anionic phenyleneethylene polymers with lipids: from membrane embedding to liposome fusion, Langmuir. 30 (2014) 10704–10711. 21] M.H. Pérez, J. Siepmann, C. Zinutti, A. Lamprecht, N. Ubrich, M. Hoffman, R. Bodmeier, P. Maincent, Non-degradable microparticles containing a [ B: Biointerfaces 147 (2016) 442–449 449 hydrophilic and/or a lipophilic drug: preparation, characterization and drug release modeling, J. Control Release. 88 (2004) 413–428. 22] E. Marin, M.I. Briceno, C. Caballero-George, Critical evalutaion of biodegradable polymers used in nanodrugs, Int. J. Nanodrugs. 8 (2013) 3071–3091. 23] A.T.R. Williams, S.A. Winfield, J.N. Miller, Relative fluorescence quantum yields using a computer-controlled luminescence spectrometer, Analyst 108 (1983) 1067–1071. 24] Z. Zhou, J. Ye, L. Chen, A. Ma, F. Zou, Simultaneous determination of ropivacaine and dexamethasone in biodegradable PLGA microspheres by HPLC, Yakugaku Zasshi. 130 (2010) 1061–1068. 25] J.L. Oliveira, E.V.R. Campos, C.M.G. Silva, T. Pasquoto, R. Lima, L.F. Fraceto, Solid lipid nanoparticles co-loaded with simazine and atrazine: preparation, characterization, and evaluation of herbicidal activity, J. Agric. Food Chem. 63 (2015) 422–432. 26] J. Asrar, Y. Ding, R.E. La Monica, L.C. Ness, Controlled release of tebuconazole from a polymer matrix microparticle: release kinetics and length of efficacy, J. Agric. Food Chem. 52 (2004) 4814–4820. 27] E.V.R. Campos, J.L. Oliveira, C.M.G. da Silva, M. Pascoli, T. Pasquoto, R. Lima, P.C. Abhilash, L.F. Fraceto, Polymeric and solid lipid nanoparticles for sustained release of carbendazim and tebuconazole in agricultural applications, Sci. Rep. 5 (2015) 13089. 28] D. Meza, E. Arias, I. Moggio, J. Romero, J.M. Mata, R.M. Jimenez-Barrera, R.F. Ziolo, O. Rodríguez, M. Ottonelli, Synthesis and photophysical and supramolecular study of �-conjugated (diethylene glycol methyl ether), Polym. Chem. 6 (2015) 1639–1648. 29] V.C.F. Mosqueira, P. Legrand, H. Pinto-Alphandary, F. Puisieux, G. Barrat, Poly(d,l-lactude) nanocapsules prepared by a solvente displacement process: influence of the composition on physicochemical and structural properties, J. Pharm. Sci. 89 (2000) 414–626. http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0060 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0065 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0070 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0075 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0080 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0085 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0090 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0095 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0100 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0105 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0110 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0115 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0120 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0125 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0130 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0135 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0140 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 http://refhub.elsevier.com/S0927-7765(16)30608-7/sbref0145 Development of stained polymeric nanocapsules loaded with model drugs: Use of a fluorescent poly(phenyleneethynylene) 1 Introduction 2 Materials and methods 2.1 Materials 2.2 Synthesis of pPy3E-sqS 2.3 Characterization of pPy3E-sqS 2.4 Preparation of the polymeric nanocapsules 2.5 Polymeric nanocapsules characterization 2.6 Release experiments 3 Results and discussion 3.1 Polymer synthesis and characterization 3.2 Staining and characterization of the nanocapsules 3.3 Colloidal stability of the stained polymeric nanocapsules 3.4 Release kinetics of model drugs 4 Conclusions and perspectives Acknowledgment Appendix A Supplementary data References