Development and Characterization of Soy Lecithin Liposome as Potential Drug Carrier Systems for Doxorubicin

Synthesis and Characterizations of SLP and DOX/SLP

The primary characterization of SLP, DOX and DOX/SLP was performed through FTIR spectra as shown in (Fig. 1A–C), respectively. Band area values were calculated using a linear baseline from 4000 to 2500 cm−1, from 2500 to 1500 cm−1, and the fingerprint from 1500 to 300 cm−1.

Fig. 1figure 1

The Fourier transform infrared (FTIR) spectra of SLP (a), Dox (B), and DOX./SLP (C)

As shown in the spectrum of SLP, the absorption band 2079 cm−1 (N = C = S), 1762 cm−1 (Cl–C = O), 1640 cm−1 (C = C), 1321 cm−1 (COO-), 1247–1206 cm−1 (C-O), 1112 cm−1 (CO–O-C), and 849 cm−1 (C–Cl) are identified. The spectrum was similar to DOX/SLP spectrum except for the appearance of bands at 3432 cm−1 (O–H), 3000–2850 cm−1 and at 1457–1383 cm−1 (C-H), 1086 cm−1 (C-F), 1018 cm−1 (C-N) (amine), and at 593 cm−1 (C–Br) [13]. Note also that the sharp peak in SLP that corresponds to the C = O disappeared in DOX/SLP, because of the presence of cholesterol chloroformate in the sample; the band Cl–C = O disappeared which means that DOX is successfully conjugated with SLP [14]. The shift of the C = C bond means there is a decrease in the frequency that marks the formation of new hydrogen bonds between components [15].

Standard Curve and Drug Loading Efficiency of DOX

For constructing the standard curve, 1 ml of DOX was diluted to different concentrations (Conc.). The absorbance of each concentration is measured by a fluorimeter. The absorbance of each sample is measured at a wavelength of 470 nm (excitation) which is plotted as a function of DOX concentration as shown in (Fig. 2). This curve will be used to calculate the drug loading efficiency (DLE) of DOX. To calculate the DLE of DOX, 1 ml of DOX/SLP was diluted in 6 ml of PBS with pH = 7.2 and then centrifuged at 12,000 rpm for 15 min at 4 °C.

Fig. 2figure 2

Standard curve of doxorubicin

Then, separate the supernatant which contains the free DOX, from that bound with SLP. A total of 0.1 ml of supernatant is diluted with 0.9 ml of PBS buffer solution, and use fluorimeter to get its absorbance. By using the standard curve, the corresponding diluted Conc. (0.03265 mg/ml) is determined from the measured absorbance value and using Eq. (2). Thus, the encapsulated drug is calculated to be 1.673 mg/ml.

The total vial Conc. is 2 mg/ml. So, the encapsulated drug is calculated to be 1.673 mg/ml. From Eq. (1), The DLE% is calculated to be 83.68%. That tells us that larger particles size has large cores, which allow more drugs to be encapsulated per particle and give slower release. Thus, as we control particle size, it provides a means of regulating drug release rates and increasing encapsulation efficiency [16]. Also, DOX is positively charged because of its protonatable amino group, so it can engage in electrostatic interactions with full or partially negatively charged groups in the SLP [17]. This all leads to increasing the EE%.

Determination of Phase Transition Temperature (Tm)

The phase transition of DOX/SLP was determined using an RF-5301PC Spectro fluorophotometer connecting with a water bath equipped with a thermocouple. This device is an important tool for the trace analysis of compounds that have functional groups that exhibit fluorescence. In this technique, the sample was diluted (0.1 ml of sample: 1.5 ml of saline), and then put the sample in the cuvette chamber which is connected to the water bath circulator. Starting from 25 to 65 °C, the intensity of fluorescence reading is recorded every 2 °C. The recorded fluorescence values are plotted as a function of temperature as shown in (Fig. 3).

Fig. 3figure 3

Transition temp., of DOX/SLP

As shown in (Fig. 3), the Tm of DOX/SLP is calculated. It is found that in DOX/SLP, the Tm increased to 47.4 °C in comparison with SLP as blank (O’Neil SD, 1982) [18]. This increase in Tm because of cholesterol in the lipid led to a decrease in the fluidity of the membrane and gives stability to liposomes and a decrease of uptake by the Reticulo-endothelial-system [19].

Drug Release by the Dialysis Bag

In the beaker, put 30 ml of NaCl saline (pH 5.5) as a buffer, and 5 cm of dialysis bag filled with 2 ml of our sample DOX/SLP. Close the bag up and down with clips and then place it over low stirring all time during the experiment. The samples were taken every 2, 4, 6, 24, and 35 h, and their absorbance at a wavelength of 470 nm is measured using the fluorimeter device. The plotted curve is the time (hours) on the X-axis and release (%) the on Y-axis to get the release curve of DOX, as shown in (Fig. 4).

Fig. 4figure 4

Release profile of DOX, by dialysis bag

Drug release from DOX/SLP was very slow until 6 h and continued to increase until the end of the experiment. By using the standard curve in the analysis, it is found that after 6 h, only 4% of the total DOX Conc. in the bag was released, while after 24 h, about 15% of the total DOX Conc. in the bag was released. Until 35 h, about 96% of the total DOX concentration in the bag was released. In other words, the release behavior of DOX from loaded SLP was significantly slower than that of DOX alone. The result showed that doxorubicin release increased and then reached a plateau region after 6 h and remained mostly constant until 24 h, and then start to increase again. This suggested the release of Dox. from SLP is too low because Dox. inside the SLP present as a precipitate that supports the Dox. stability inside the SLP [20]. This release time increase helps to keep DOX/SLP in circulation for a longer time with min. amount of release in blood circulation. That helps in decreasing the side effects of DOX, as a drug on healthy tissue [21]. As the encapsulation efficiency is high, the stability of liposomes is high (size distribution, suitable preparation methods, usage of high-quality lipids, suitable preparation method, and storage conditions), all of this preventing drug leakage [22].

The Optimal Formulation of SLP and DOX/SLP

The size and zeta potential of SLP was measured to be 282 nm and −26.9 mV respectively, while the measured values of DOX/SLP were 342 nm and −22.3 mV respectively. As shown in (Fig. 5), there is an increase in size that can be explained by increasing the attractive force between SLP and DOX leading to increasing the size [23]. That size affects the amount of drug loading, which pivotally determines the pharmacokinetics and pharmacodynamics of the drugs in circulation [24]. As we know that zeta potential is the total surface charge and the stability of SLP or SLP/DOX. When liposomes have charge means, repulsive forces increase and the medium becomes stable. Zeta potential lesser than −30 mV or greater than 30 mV is considered to be more stable [25]. So, the magnitude of the zeta potential increase from −26.9 to −22.3 mV gives a sign of the potential stability of the sample, by increasing the repulsion between particles. This zeta potential has the possibility to improve biological performance by circumventing surface charge-related toxicities [26].

Fig. 5figure 5

DLS image size, of SLP (A) and DOX/SLP (B)

Transmit Electronic Microscope (TEM)

The morphology of both samples in detail was further confirmed by TEM. This technique can give highlight structural changes in any material. The samples were prepared by placing a drop from each sample on carbon-coated copper placed on filter paper. Let dry for a few seconds, then place a drop of transmittance-negative stain on it, and leave it to air-dry before imaging. As shown in (Fig. 6), TEM verified the presence of liposomes in round shape with multi-lamellar, 200 nm in width in the SLP sample while in DOX/SLP, the sample was round, dark in color because of DOX, and has around 320 nm in width. The TEM images showed that both SLP and DOX/SLP were spherical in morphology, and the liposomal system possessed a size range that fell into the therapeutic-potential range [27].

Fig. 6figure 6

TEM images, of SLP (a) and DOX/SLP (b)

Cell Line Cytotoxicity

Cytotoxicity assays were performed to evaluate the biocompatibility of synthesized DOX/SLP as drug delivery systems in the normal cell line (Vero) (Table 1) and the human breast cancer cell line Mcf-7 in both low and high DOX and DOX/SLP concentrations (Tables 2 and 3) respectively, followed by Fig. 7 which describes the histograms ± SD of all analyses of cell viability. Results were expressed as the percentage of cell proliferation, compared with 0.1% DMSO control and were calculated from Eq. (3) as follows [28]:

Table 1 Chemo-sensitivity testing in a normal cell line (Vero) using MTT assay (by single-fold dimension). This table shows the results were expressed as IC50, i.e., the concentration of cytotoxic drug that reduces cell viability by 50% relative to the controlTable 2 Chemo-sensitivity testing in Mcf-7 cell lines (low concentration) using MTT assay (by double-fold dimension). This table shows the results were expressed as IC50, i.e., the concentration of cytotoxic drug that reduces cell viability by 50% relative to the controlTable 3 Chemo-sensitivity testing in Mcf-7 cell lines (high concentration) using MTT assay (by double-fold dimension). This table shows the results were expressed as IC50, i.e., the concentration of cytotoxic drug that reduces cell viability by 50% relative to the controlFig. 7figure 7

Cell viability of A Vero as a normal cell exposed to 100, 50, 25, 12.5, 6.25, and 3.125 µg/ml, B Mcf-7 cells exposed to 100, 50, 25, 12.5, 6.25, and 3.125 µg/ml, and C Mcf-7 cells exposed to 250, 125, 62.5, 31.25, 15.62, and 7.812 µg/ml of DOX (orange line) and DOX/SLP (gray line) on all

$$Viability\;\left(\%\right)=\frac\times100$$

(3)

As shown in (Table 1, Fig. 7A), the normal Vero cell line was exposed to 100, 50, 25, 12.5, 6.25, and 3.125 µg/ml of both DOX and DOX/SLP separately. When comparing cell viability at all conc. of DOX/SLP to cell viability of DOX only, a difference in cell viability was observed and cell viability decreased from low to high conc. in DOX/SLP records, while there is approximate stability of cell viability in lower conc. in DOX results. IC50 ± SD of both DOX and DOX/SLP was 41.75 ± 0.77 and 11.08 ± 0.07, respectively. This difference possibly returns to the mechanism of release of DOX from SLP over the incubation time [29].

As shown in (Table 2, Fig. 7B), cell viability assay can determine the effect of drug candidates on cells and be used to optimize the cell culture conditions. As shown DOX/SLP has an inhibition effect on cell growth [30]. This data represent the effect of DOX and DOX/SLP on Mcf-7 cell lines by using low sample conc. 100, 50, 25, 12.5, 6.25, and 3.125 µg/ml. These data showed that the conc. of the drug that needs to kill 50% of cancer cells is low in DOX/SLP rather than DOX only. When DOX/SLP reach the tumor site, they released DOX which is capable of diffusing through the tumor cell membrane to the nucleus which contains DNA of the tumor cell to block topoisomerase II and cause cell death [31]. IC50 ± SD of both DOX and DOX/SLP was 6.01 ± 0.28 and 4.81 ± 0.07. This indicates that liposomes form a shell around DOX, which led to reduced DOX release as indicated in the drug release profile described above, and the amount of DOX released increases after 72 h of injection [32].

The last one is shown in (Table 3, Fig. 7C), the effect of higher sample conc. 250, 125, 62.5, 31.25, 15.62, 7.81 µg/ml on Mcf-7 cell line. The same effect of DOX/SLP in higher conc. on higher conc. especially at 31.25, 15.62, and 7.81 µg/ml, is clearly shown a great difference in cell viability rather than DOX only. When DOX/SLP reaches the nuclease, a large amount of DOX diffuses into the tumors’ nucleus. Overall conc. IC50 ± SD of DOX. and DOX/SLP was 41.02 ± 0.66 and 27.72 ± 0.96, respectively. This proves that the encapsulation of DOX is high and SLP controlled in DOX release. So, it can be concluded that DOX/SLP are biocompatible, bioavailable, and appropriate for drug delivery systems [33].

The cell toxicity caused due to the action of a chemotherapeutic agent on Mcf-7 cells is described in After Analysis; it is clearly shown that at higher concentrations (250, 125, and 62.5 µg/ml), there is a low sample toxicity effect rather than free DOX. The smaller value of IC50 of DOX/SLP in all conc. means that the amount of drug-loaded liposome that needs to kill 50% of cancer cells is less than the amount of free drug, which concludes that much lower concentrations of drug load by liposomes will have as a great effect on cells as higher concentrations of free drugs which will greatly reduce the side effects of using larger doses of chemotherapeutic drugs [34].

There are some images that indicate the morphological changes on different cell lines, represented as follows: control cell line, after adding free DOX and after adding the same conc. from DOX/SLP. As shown in Figs. 8, 9, and 10, there is a clear difference between the effect of free DOX and DOX/SLP on the cancer cells.

Fig. 8figure 8

Morphology of Vero cells: a control Vero (A′), the effect of free DOX with concentration (6.25 µg/ml) (B′), and the effect of DOX/SLP with concentration (6.25 µg/ml) (C′)

Fig. 9figure 9

Morphology of Mcf-7 cells at low conc.: a control Mcf-7 (A), the effect of free DOX with concentration (6.25 µg/ml) (B), and the effect of DOX/SLP with concentration (6.25 µg/ml) (C)

Fig. 10figure 10

Morphology of Mcf-7 cells at high conc.: a control Mcf-7 (a), the effect of free DOX with concentration (31.25 µg/ml) (b), and effect of DOX/SLP with concentration (31.25 µg/ml) (c)

According to published data in 2013, by comparing the therapeutic efficacy of Doxil (commercial doxorubicin-loaded-liposomes) prepared from animal origin source, the cytotoxicity as presented by its IC50 of free DOX, DOX/SLP, and Doxil is shown in (Fig. 11). The IC50 values of DOX, DOX/SLP, and Doxil are calculated to be 41.02 ± 0.66 µg/ml, 27.72 ± 0.96 µg/ml, and 32.2 ± 3.6 µg/ml respectively [35]. The smaller value of IC50 of DOX/SLP against free DOX and Doxil indicates the more therapeutic efficiency of DOX/SLP as a potential and novel chemotherapeutic confirmation concerning other chemotherapeutic drugs, such as free DOX and Doxil.

Fig. 11figure 11

The cytotoxicity as indicated by its IC50 of DOX, DOX/SLP, and Doxil

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