7-MEOTA was synthesized by following the known and optimized procedure [46, 47] in the laboratory (Scheme 1) with a purity of higher than 95%. The chemical structure of 7-MEOTA was characterized by means of 1HNMR, 13CNMR and FT-IR techniques. Spectral data were in good agreement with the literature. Condensation reaction of 4-methoxyaniline 1 with ethyl 2-oxocyclohexanecarboxylate 2 in presence of p-toluenesulfonic acid (pTsOH) as an acidic catalyst and refluxing toluene yielded 7-methoxy-1,2,3,4-tetrahydroacridin-10H-9-one 3 up to 80% yield. Reaction of phosphorus oxychloride (POCl3) with 3 gave 9-chloro-7-methoxy-1,2,3,4-tetrahydroacridine 4 with excellent yield. The compound 4 is converted into 7-MEOTA with ammonium carbonate in phenol at 120°C.
Spectral data of 7-MEOTA (9-Amino-7-methoxy-1,2,3,4-tetrahydroacridine): m.p.= 211–215°C; FT-IR (KBr, υ/cm− 1): 3458.70, 2932.54, 1650.98, 1573.71, 1501.89, 1460.29, 1379.65, 1234.99, 1032.39; 1HNMR (500 MHz, CD3Cl) δ: 1.89 (m, 4H), 2.57 (t, 3J = 11.5 Hz, 2H), 2.98 (t, 3J = 11 Hz, 2H), 3.79 (s, 3H, OCH3), 4.68 (s, 2H, NH2), 6.95 (s, 1H), 7.22 (d, 3J = 10 Hz, 1H), 7.81 (d, 3J = 10 Hz, 1H); 13CNMR (500 MHz, CD3Cl) δ: 22.99, 23.08, 24.01, 34.03, 55.58, 98.90, 110.94, 117.73, 120.46, 130.54, 142.66, 145.66, 156.28 ppm.
The amounts of 7-MEOTA present in the PCL nanocapsules suspensions were determined by UV–Vis technique at λmax = 246 nm and liquid chromatography-tandem mass spectrometry, after filtration through a 0.22-µm Millipore membrane, 30 kDa. According to the concentration and chromatographs of the standard drug samples and analysis of nanocapsules solution, the amount of free 7-MEOTA in suspensions has been obtained as 35%. Thus, 65% of the initial drug amount has been associated with PCL nanocapsules. Examining the PCL suspensions after one month, no significant change is observed in the initial free drug concentration (Fig. 1). It should be mentioned that the results extracted from UV-visible method were also in line with LC-MS/MS data.
The stabilities of the 7-MEOTA@PCL suspensions were evaluated using measurements of their diameter, polydispersity index, and zeta potential, as a function of time over a 40-day period (0, 10, 20, 30 and 40 days). The average sizes of the 7-MEOTA@PCL nanocapsules were 237 nm (Fig. 2a). The values represent the average of three experiments performed at room temperature. No significant difference was observed in the nanocapsule size throughout the 40 days of the experiment. Size distribution of the 7-MEOTA@PCL nanocapsules after 10 days was shown in Fig. 2b. As can be seen, a narrow particle size distribution of 7-MEOTA@PCL nanocapsules was obtained with the interfacial deposition of pre-formed polymer method (experimental section).
10, 20, 30 and 40 days) (a) and size distributions of the 7-MEOTA@PCL nanocapsules after 10 days (b).
The polydispersity index (PI) in different directions which indicates the dispersion of the particle sizes, was used to evaluate the stability and uniformity. The values of PI of 7-MEOTA@PCL nanocapsules at the beginning of the period, was determined as 0.31. The PI values of smaller than 0.5 is ideal for colloidal suspension. Good particle homogeneity was observed from the polydispersity results. After 40 days, the 7-MEOTA@PCL nanocapsules remained stable, but PI value for the nanocapsules increased slightly that showing greater instability, due to aggregation of the nanocapsules (Fig. 3).
The value of zeta potential of 7-MEOTA@PCL nanocapsules at the beginning of the period was estimated as -21.2 mV. This potential shows the charge present on the particle surface. The zeta potential in the nanocapsules is negative due to the presence of carboxylic groups (-COO−) in the PCL chemical structure, since PCL is a polyester. The zeta potential value with an amount of ± 30 mV is considered to be good for the suspension. In addition, the zeta potential of the 7-MEOTA@PCL nanocapsules remains negative after 40 days, pointing out to their complete stability and lack of any aggregation. This is mainly due to the fact that the particles charge is sufficient so that electron repulsion prevents the particle aggregation.
The FT-IR spectra of 7-MEOTA, empty PCL, and 7-MEOTA@PCL nanocapsules are plotted in Fig. 5. Comparing the absorption frequencies in the FT-IR spectra is considered as a good benchmark for interaction investigating between the 7-MEOTA and PCL nanocapsules interaction. As can be seen from Fig. 5a, the bands at 3458 and 3320 cm− 1 correspond to stretching of the N-H bond present in the primary amine group of the 7-MEOTA structure, while
bands at 2826–3063 cm− 1 are related to stretching of the alkyl (sp3) and aromatic (sp2) C-H bonds. Aromatic C = C stretching frequencies of 7-MEOTA can be observed at 1501–1600 cm− 1. Bending absorption of methylene and methyl groups occur around 1460 and 1379 cm− 1, respectively. The absorption frequencies of the C–N bonds also appear at 1032–1235 cm− 1. Furthermore, the peak observed at 1650 cm− 1 is related to bending frequency of N–H bond in the 7-MEOTA structure.
The FT-IR spectra of empty PCL indicate the absorption frequencies at 3436 cm− 1 (O-H bond stretching), 2949 cm− 1 (C-H bond stretching) and a characteristic stretching absorption at 1731 cm− 1 for carbonyl group of ester. The spectrum of the 7-MEOTA@PCL is almost similar to that of the empty PCL. Due to very small amount of the 7-MEOTA with respect to PCL nanocapsules, the frequencies related to the nanocapsules usually cover the absorption ones associated with the 7-MEOTA. The formation of 7-MEOTA@PCL nanocapsules can be confirmed by FT-IR spectra as absorption frequencies were shifted after interaction of PCL nanocapsules with 7-MEOTA (Fig. 5b and 5c).
The morphological characteristics of the 7-MEOTA@PCL nanocapsules were studied using transmission electron microscopy (Fig. 6a) and scanning electron microscopy (Fig. 6b). The TEM images showed that the 7-MEOTA@PCL nanocapsules were nearly spherical with uniform size distribution, and there was no tendency for aggregation. The average size was in the range 200–300 nm. The SEM micrograph confirmed solid dense nanocapsules with fully polymeric structure (Fig. 6b).
The release profile of the encapsulated 7-MEOTA from the PCL nanocapsules is shown in Fig. 7. The profile shows the release of 7-MEOTA in 3 steps with different slopes. At first step, the initial explosive release of the drug occurs and about 50% of the initially encapsulated 7-MEOTA was released from the PCL nanocapsules after 1 h. The initial fast release of 7-MEOTA observed into buffer probably due to the dissolution of 7-MEOTA attached to the surface of the PCL nanocapsules and the beginning of the drug release process due to the water penetration into PCL nanocapsules and escape of the drug through the concentration gradient. The second stage includes a range of one hour after the start to 3 hours. The slope of the chart is reduced at this step and it can be seen the gradual release of the drug from inside the PCL nanocapsules. In the last step, the slope of the diagram is almost fixed. The release percentage of around 90% was obtained after 24 h, while the release of the drug reached 95% after 72 h.
In order to evaluate the release mechanism the drug from PCL nanocapsules, the drug release results were matched with the mathematical model proposed by Korsmeyer and Peppas [48, 49], described by: