A rhodamine B-labeled triglyceride (product 1) was obtained in order to prepare fluorescent nanocapsules with different properties, such as anionic or cationic surfaces, achieved by changing the polymer used to prepare the nanocarrier. Fluorescent LNC were also prepared.The RhoB carboxyl group was activated by a carbodiimide. This intermediate product reacted with the hydroxyl groups of ricinolein, contained in the castor oil, to produce an ester (product 1) (Figure 1). The fluorescent-labeled product 1 was purified in a preparative chromatographic column. The TLC (Figure 2) image, revealed with UV light, indicated that a fluorescent product was obtained without contamination of the unbound rhodamine B. Thus, the purification process was effective in removing molecules of rhodamine B that did not react with the ricinolein from the crude fluorescent product 1. The presence of free rhodamine B in the final product could lead to release of the fluorescence from the nanocapsule and thus unreliable results. The several spots observed for the purified fluorescent product 1 were expected since castor oil is a mixture of triglycerides and also because the rhodamine B molecule can react with one, two, or three of the hydroxyl groups presented in the ricinolein residue, which could result in products with different polarities.
The FTIR and 1H-NMR spectra (Figure 3 and Additional file 1: Figure S1B) showed that the main structure of the raw castor oil was maintained after the reaction. No band characteristic of carboxylic acid was observed on the FTIR spectrum of the purified product (Figure 3), and the signal with a chemical shift of 2.3, characteristic of the hydrogen atoms of an ester, was maintained (Additional file 1: Figure S1B). This suggests that no hydrolysis of the ester bound occurred. 1H-NMR spectrum of the fluorescent product 1 showed signals with chemicals shifts higher than 5.8 and an AB system corresponding to the hydrogen atoms of the aromatic ring of rhodamine B residue. However, as previously reported, the sensitivity of FTIR and 1H-NMR techniques can be not sufficient to detect some functional groups or the protons of the dye due to their small contribution compared to the contribution of the functions and hydrogen atoms of the oil residue [12, 28]. Up to this point, the results (TLC, FTIR, and 1H-NMR) indicate that the functional carboxylic group of rhodamine B was bound to the ricinolein presented in the castor oil and that a fluorescent oily product was obtained presenting good purity regarding the presence of unbound rhodamine B.
UV-vis and fluorescence spectroscopy showed that the product 1 obtained presents maximum absorption (λmax-ab = 519 nm) in the green region of the optical spectrum and maximum emission (Figure 4) in the yellow-orange region (567 nm). The results for the SEC analysis of the purified product 1 were consistent with the values obtained for the raw castor oil, demonstrating that the hydrodynamic volume and the size chain distribution were not modified after rhodamine B coupling to the product. The quantitative analysis of the amount of rhodamine B bound to the product indicated a concentration of bound dye of 0.517 ± 0.096 μmol per g of fluorescent oily product (n = 3). This corresponds to 1 rhodamine residue for 1,150 molecules of the product.
The rhodamine-labeled triglyceride was used to prepare fluorescent NC formulations with Eudragit RS100 or Eudragit S100, providing cationic and anionic particles, respectively. Fluorescent LNC were also prepared with the rhodamine-labeled product using poly(ϵ-caprolactone) as the polymer. The liquid portion of the nanocapsule core was composed of fluorescent triglyceride (10%) and CCT (90%) (Table 1). In the LNC-PCL formulations, the liquid portion was 160 μL/10 mL of suspension corresponding to approximately 1.52 mg of fluorescent product per mL of suspension, while in NC-RS100 and in NC-S100, the liquid portion was 333 μL/10 mL of suspension corresponding to approximately 3.15 mg of fluorescent product per mL of suspension. It is important to note that the amount of rhodamine-labeled triglyceride can be increased or decreased, according to the needs of the study. The pH of the nanocapsule formulations (Table 1) was slightly acid and similar to the values previously reported for formulations prepared without the fluorescent-labeled oil [26, 29]. The size distribution profiles (Figure 5) and the D4.3, SPAN, z-average, PDI, and zeta potential values for the formulations containing the fluorescent product 1 (Table 1) did not differ considerably from those observed for non-fluorescent formulations [25–27].
The zeta potential values for the formulations prepared with the fluorescent product 1 (Table 1) showed values approximately closed to those previously reported for the similar formulations prepared without the dye-labeled oil [25–27]. The electrokinetic behavior of colloids is related to the movement of ionic solutions near charged interfaces . The carboxylic acids, as pendant groups in Eudragit S100 or as terminal groups in PCL116, are in an acid-base balance at the particle-water interface producing carboxylate functions that react with NaCl forming the electrical double layer responsible for the eletrokinetic behavior of NC-S100 and LNC-PCL. On the other hand, the NC-RS has a polymer wall of poly(ethyl acrylate-co-methyl methacrylate-co-trimethylammonioethyl methacrylate chloride), whose monomer units are at 1:2:0.1 proportions. In this way, the trimethylammonioethyl moiety has a quaternary nitrogen giving to the particle-water interface a positive charge. The electrokinetic properties of NC-RS are related to the positive surface potential that those nanocapsules present after dilution in 10 mmol L-1 NaCl aqueous solution. Considering that all formulations contain polysorbate 80, the mechanism of stabilization of those colloids is not exclusively based on the electrical repulsion of the particles since the steric hindrance effect of the surfactant plays an important role [31–33]. Then, even though the zeta potential values are near zero for all formulations, the colloidal turbid solutions have an adequate kinetic stability for the purpose of drug delivery. The NC-RS100 and NC-S100 formulations presented higher concentrations of particles (approximately 1.7-fold and 1.4-fold, respectively) than LNC-PCL (P < 0.05). This result was expected since the volumetric fraction of the dispersed phase in these formulations is higher than that of LNC-PCL, and the z-average values obtained for each formulation were similar .
The fluorescence spectroscopy analysis of the fluorescent nanocapsules and fluorescent lipid-core nanocapsules showed that the fluorescence property is maintained after the preparation of these formulations (Figure 6). The difference in the fluorescence intensity on comparing the NC-RS100 and NC-S100 formulations with LNC-PCL was expected since the concentration of fluorescent product in these formulations varied (approximately 3.15 mg of product 1/mL of suspension for NC-RS100 and NC-S100 and approximately 1.52 mg of product 1/mL of suspension for LNC-PCL) (Figure 6). In the undiluted/unextracted samples of the formulations, it was seen that the bathochromic (7 nm) shift for the λmax-em value in the emission spectrum of the NC-S100-1 formulation was accompanied by a hyperchromic shift (52 a.u.) when compared to the NC-RS100-1 formulation, which contains the same quantity of fluorescent product, probably due to protonation of the amino group of rhodamine B, as the pH of this formulation was the lowest among the formulations (3.50 ± 0.09). As previously reported, rhodamine B has an equilibrium of isoforms, lactonic and the zwitterionic isomers . The zwitterion isomer can be protonated more than once due to the presence of two amino groups . A hypochromic shift was observed in the emission spectra of the undiluted/unextracted samples of the LNC-PCL-1 (114 a.u.), NC-RS100-1 (230 a.u.), and NC-S100-1 (178 a.u.) formulations compared to the spectrum of the solutions containing the same quantity of the CCT/fluorescent oily product mixture in ACN [solution 1 (1.52 mg/mL) and solution 2 (3.15 mg/mL)] (Figure 6A,B). Unsurprisingly, in the case of the samples containing the CCT/fluorescent oily product mixture (Figure 6C,D), the results for the fluorescence intensity of the diluted/extracted samples of the formulations showed greater similarity when compared to the undiluted/unextracted samples. The previously observed hypochromic shift did not occur and a small hyperchromic shift occurred, especially for NC-RS100-2 (24 a.u.) and NC-S100-2 (27 a.u.). Therefore, these changes in the fluorescence intensity of the undiluted/unextracted samples are probably related to the volume fraction of particles in the dispersed phase of the formulation leading to phenomena such as the inner filter effect, where the presence of other compounds can partially absorb the emission energy, and they were not sufficiently reduced even with the use of a triangular cuvette [35, 36].
To demonstrate the applicability of the synthesized fluorescent triglyceride (product 1) to the identification of particles containing this compound in image studies, a cell uptake study was performed. It was possible to observe red fluorescence in the cells treated with the fluorescent nanoparticles (Figure 7). The red fluorescence was very close to the cell nucleus suggesting that the particles are located inside the cells. Martins and co-workers  have reported the uptake of solid lipid nanoparticles (SLN) stabilized with polysorbate 80 by THP1-derived macrophages. The authors loaded the SLN with a green fluorescent dye and evaluated the particle uptake by fluorescence microscopy. In a recent study, our research group demonstrated the uptake of LNC-PCL, also stabilized with polysorbate 80, by macrophages isolated from BALB/c mice . In this case, the LNC-PCL particles were prepared with the polymer chemically bound to rhodamine-B and non-labeled oil. The results reported herein reinforce these findings and can demonstrate the applicability of the use of the fluorescent triglyceride to localize particles in biological studies with the advantage of allowing the development of tracking systems with surfaces exhibiting a variety of chemical natures. In a forthcoming publication, the applicability of this product to tracking particle skin penetration and also particle uptake by skin cells, considering the influence of the particle surface properties, will be demonstrated.
Recently, in an in vivo study with rats implanted with glioma tumors, it was showed that, after 10 days of treatment, the group of animals treated with indomethacin loaded in LNC (IndOH-LNC) particles presented a higher concentration of the drug in the cerebral tissue and, more specifically, in the tumor hemisphere compared to the group which received the free drug . The tumor size of the groups treated with IndOH-LNC  or trans-resveratrol loaded in LNC (t-resv-LNC)  particles was significantly reduced when compared to the groups treated with the free drug. A similar profile of higher drug concentration in the brain compared to the free drug was observed in a biodistribution study in rats treated with trans-resveratrol or t-resv-LNC particles . Based on these findings, it is suggested that LNC particles are able to target the drug to the brain tissue and reduce the tumor size. The synthesis of fluorescent materials for the preparation of fluorescent dye-labeled nanocapsules, such as the fluorescent polymer  and the fluorescent triglyceride, product 1 (as reported herein), could also be useful for tracking the pathway of the LNC particles and/or their uptake in cells, for instance, in experiments similar to those cited here. Therefore, the labeled nanoparticles may be used to find the final destiny of the particles after in vitro and in vivo treatments.