In this study, what were used are polycaprolactone (PCL) (Mn = 10,000 g/mol), Pluronic® F-68 (Mn = 8400 g/mol), acetone, ethanol, chloroform, and ethyl acetate P.A. All were obtained from Sigma-Aldrich®. Flaxseed oil was extracted in the laboratory.

For the extraction of flaxseed oil, the Soxhlet method was used. The seeds were crushed with the aid of a blender, turning them into flour, and then 10 g of the flour was weighed and stored in a paper filter to be subsequently placed inside the Soxhlet extraction chamber. With the apparatus set up, the extractions were made with 10 cycles using ethanol, chloroform, and ethyl acetate/ethanol (3:1) in each extraction. In the end, the solvents were evaporated in a rotary evaporator.

To carry out the encapsulation of flaxseed oil, it was necessary to prepare two solutions in advance, one being the aqueous phase and the other the organic phase. The aqueous phase contained 100 ml distilled water and 0.12 g Pluronic®, and the organic phase contained 50 ml acetone, 0.20 g PCL polymer, and 0.10 g of the previously extracted flaxseed oil.

The aqueous phase was immediately subjected to magnetic stirring. Regarding the organic phase, the oil was initially solubilized in acetone using an ultrasonic tip sonicator for one minute with 40% power and a 4 mm micro tip. Then the polymer was added, and the solution was kept under magnetic stirring until solubilization of the PCL.

The next step is the nanoprecipitation, the organic solution was poured into the aqueous solution using a glass funnel for liquid in a continuous way. In the end, the emulsion was kept for three days under magnetic stirring at room temperature to the solvent evaporation. After this period, the sample was taken to the freezer for 6 hours at −62˚C and finally to the lyophilizer under the pressure of 2000 µHg and −47˚C. The methodology used in this project was adapted from the literature [18].

The magnetic resonance spectra for the extractions in the different solvents used were obtained, however, they all presented the same signal profile. Thus, only the results obtained for one of the extractions will be presented. Along with NMR, FTIR and the study of the literature are going to be used [19] [20] [21].

Figure 1 presents the ¹H spectrum for brown flaxseed oil extracted in ethyl acetate/ethanol (3:1) and Table 1 lists each signal obtained in the spectrum with its chemical shift.

¹ [20]; ² [19].

The data obtained from the hydrogen and carbon spectra as well as the literature data indicate that there is a mixture of fatty acid esters bonded to the three carbons of glycerol [19] as shown in Figure 2. The carbons 1 and 3 of the glycerol are linked to the same or very similar molecules, which keeps the symmetry in the molecule.

In the ¹H NMR spectrum, two methyl signals can be highlighted, one referring to the α-linolenic acid ester (signal B) and the other of the other esters including linoleic acid (signal A), the methyl B signal is more deshielded due to its proximity to a double bond which does not occur with signal A.

Another important sign is the doublet of doublets at the center (H sign) this refers to the hydrogens of the CH₂ groups present in glycerol. The signs C, D, E, F, and G refer to the CH₂ groups present in the chain of these esters, with F being the hydrogens closest to the carbonyl and G being those located between the double bonds, the sign J represents the hydrogens of the double bonds. The description of these signals is summarized in Table 1.

In addition to the ¹H spectrum, the ¹³C and APT spectra were obtained (Figure 3 and Figure 4, respectively). The APT spectrum is an important tool to distinguish the types of carbons present, it is possible to observe the methyl groups and CH groups in an inverted way on the spectrum and thus discriminate them from the CH₂ groups and carbons that do not have hydrogen bonded.

In these spectra, it is possible to observe that there are two signals referring to a glycerol molecule. The first at 62.62 ppm for CH₂ and the second at 69.68 ppm

for CH, this can be explained by the symmetry of the molecule, causing the two CH₂ signals to fall into the same chemical shift. This issue is reinforced by the signs of the ester carbonyls at 173.36 ppm and 173.77 ppm, the methyls (CH₃) at 14.65 ppm and 14.81 ppm and the α CH₂ at 34.54 ppm and 34.71 ppm which has, as can be seen in the enlargements, two signs each (Figure 3).

In order to aid the characterization of the flaxseed oil, the HSQC (Single Quantum Coherence Spectroscopy) and COSY (Correlation Spectroscopy) spectra were also obtained, Figure 5 and Figure 6 show these spectra. The first spectrum correlates the hydrogen signals with the carbons which are directly bonded and the second correlates ¹H signals with other ¹H signals up to three bonds, therefore on adjacent carbons.

In the first part of the spectrum (signals from A to G in the ¹H phase), it is possible to observe that these are the hydrogens present in CH₃ and CH₂ correlated with their respective carbons. The most notable of these correlations is between the hydrogens and carbons of the methyls (signals A and B in the ¹H spectrum) and the ¹H α with α carbon (F signal) as shown in Figure 5.

In the second part of the spectrum, the doublet of doublets can be observed at the center of the spectrum (signal H), in the hydrogen phase, these signals can be correlated with the CH₂ carbons of glycerol. Signal I refers to the central hydrogen of glycerol and although it is overlap on sign J, it is possible to see its correlation with the respective carbon. Guillén and Uriarte (2012) described this CH hydrogen signal around 5.20 to 5.26 as shown in Table 1.

Finally, the third part of the spectrum shows the correlations between the unsaturation hydrogens (signal J) with their carbons. The last signal that appears in

the carbon phase, in which there is no correlation on the spectrum is the carbons of the ester carbonyls, this signal is also doubled in the enlargement of the 13C spectrum.

Regarding the COSY spectrum (Figure 6) in the first part, it is possible to highlight the methyl A signals with the CH₂ groups that are not close to the unsaturation (C signal), this last signal also correlates to the E signal that refers to neighboring hydrogens of the double bonds (pink arrows on the spectrum). The E signal correlates with the B in the case of the α-linolenic acid ester molecule due to unsaturation near the chain ends.

It is also possible to observe the correlation between the F and D signals, respectively, the α and β hydrogens of the esters chain. Then we have the correlation between H and I (overlapped on the J signal), that is, the hydrogens of the CH₂ groups of the glycerol with its central hydrogen. Finally, it is possible to correlate the J signal (¹H of the unsaturation) with the signals G and E CH₂ near the unsaturation carbons. Table 2 summarizes all the signals handled so far.

The FTIR analysis confirms the presence of unsaturated fatty acid ester due to the presence of bands of 3010 cm⁻¹, 1739 cm⁻¹, 1647 cm⁻¹ and 1242 cm⁻¹, these and the other bands are also reported in other research such as in Bãlãnucã et al. (2014). The band at 1739 cm⁻¹ refers to the stretching of the ester carbonyl and the band at 1242 cm⁻¹ to the stretching of the CO bond. Moreover, the bands at 3010 cm⁻¹ and 1647 cm⁻¹ are attributed to the double bonds present in the chains as can be seen in Table 3 and Figure 7.

DLS technique was performed aiming to determine particle size and polydispersity (PDI) after the oil encapsulation. Table 4 shows the mean diameter size and PDI values for the analysis performed at the end of lyophilization.

The mean diameter obtained in the analysis indicates that the particles formed are microparticles. The PDI indicates the homogeneity of the system in relation

to the particle size distribution. Thus, the smaller the PDI value, the more monodisperse the sample (this value varies between 0 and 1).

In this way, for the analysis in question, it is possible to observe that the material after encapsulation has a wider size distribution, which may be due to the collapse of the sample during the solidification and lyophilization processes [18].

During the freeze-drying process, also called lyophilization, there was an oscillation of pressure and temperature during the sublimation of the solvent, causing the sample to be solidified again in the freezer. Furthermore, a cryoprotectant was not used, so some particles may have agglomerated and reorganized, generating a mean diameter with a high standard deviation and a polydispersity around 0.674 indicating a greater polydispersion, however for this methodology there are cases in the literature with better results such as Tavares et al. (2017).

Thus, even with the problems faced, the project obtained PCL microparticles through the nanoprecipitation process, but it is important to have greater control over the parameters, especially in the freeze-drying step.

The X-ray diffraction analyses for the analyzed materials were obtained in the 2θ range between 10˚ and 60˚, as outside this range, no significant diffraction maxima were observed for the analyzed polymers or for the sample after encapsulation. Table 5 shows the angles obtained in the analyses performed.

Figure 8 shows the diffractogram of pure PCL, it is possible to observe the diffraction maxima of this polymer at 2θ = 21.7˚ and 23.9˚, this is due to the semi-crystalline structure of this polymer and corresponds to the diffraction maxima described in the literature, corresponding to the planes (110) and (200) of the orthorhombic crystal structure, respectively [22]. Other diffraction maxima can also be observed above 2θ = 30˚, these correspond to less important crystalline domains of the polymer used in the analysis.

In relation to Pluronic® F-68 (Figure 9), diffraction maxima at 2θ = 19.4˚ and 23.4˚ were observed, also similar to those observed in the literature [23].

Regarding the material after encapsulation, in Figure 10, the sample showed three diffraction maxima, at 2θ = 20.4˚, 22.6˚, and 24.8˚. The first may be associated with Pluronic® F-68, the second with PCL polymer, and the third with an overlap of the PCL and Pluronic® F-68 diffraction maxima.

Another factor to be considered is that during the analyses, it was necessary to press the material into the sample holder, which caused it to change its visual appearance, that is, it was no longer a white powder and became a more compact mass that is difficult to be worked on and with slightly different coloration, this suggests that at that time there was an oil release and this, as well as Pluronic® F-68, affected the crystallinity of the PCL. This can be highlighted when calculating the degree of crystallinity of the polymers and the sample, the PCL had 30% crystallinity, the Pluronic® F-68 18%, and the sample 27%. Thus, the oil reduced the crystallinity of the PCL in the sample.

In order to confirm whether or not the oil was encapsulated, pulse sequences MSE-FID (Magic-sandwich echo-Free induction decay) were used. The data of the material after encapsulation, PCL, and Pluronic® F-68 can be analyzed in Table 6.

In the MSE-FID sequence, a longer T₂* time indicates greater mobility of the phase in question. Then, using the data in Table 6, it can be stated that the flaxseed oil was encapsulated since the sample presented an increase in time T₂* compared to the PCL values, this means that there was an increase in the mobility of the phases, an indication that the oil was encapsulated. This increase can also be observed when comparing the T₂* of the intermediary and rigid phases of Pluronic® and the material after encapsulation.

Besides the MSE-FID sequence, the FTIR technique was also used to confirm that the oil was encapsulated. Figure 11 shows the FTIR spectrum of the material after encapsulation, in this spectrum, there are bands similar to those of the PCL polymer. Nevertheless, the bands of Pluronic® F-68 and the characteristic bands of the oil are not observed thus being another indication that the flaxseed oil was encapsulated.

In the microencapsulate, the band at 3444 cm⁻¹ is observed, similar to that present in the PCL spectrum. Moreover, the ester band at 1730 cm⁻¹ is characteristic of the polymer, remembering that in the oil spectrum there is also a band of the same type, but in 1739 cm⁻¹.

In addition to these bands those that occur in 1288 cm⁻¹, 1242 cm⁻¹, and 1177 cm⁻¹ stand out, these are also polymer bands, since they do not resemble the flaxseed oil ones that occur in the same region, but with those that are present in the PCL spectrum.