Chitosan nanoparticles containing limonene and limonene-rich essential oils: potential phytotherapy agents for the treatment of melanoma and breast cancers

Background Melanoma and breast cancers are two common cancers worldwide. Due to the side effects of chemotherapy drugs and the occurring resistance against them, the development of green drugs has been received more attention. Methods The anticancer effects of three essential oils from the Citrus family and their identified major constituents (limonene) were first investigated against melanoma and breast cancer cell lines (A-375 and MDA-MB-468). By preparing chitosan nanoparticles containing them, an attempt was then made to improve their effectiveness. Results Chitosan nanoparticles containing Citrus sinensis and Citrus limon essential oils with IC50s of 0.03 and 0.124 μg/mL on A-375 cells, and 23.65 and 40.32 μg/mL on MDA-MB-468 showed distinct anticancer efficacies. Conclusion The prepared formulations could thus be considered as green anticancer agents in complementary medicine and therapies.


Background
Breast cancer is one of the most common cancer, followed by prostate, lung, and colon cancers worldwide [1]. Breast cancer is the commonest life-threatening malignancy, causing 14% of all cancer-related fatalities among women [2,3]. Skin cancer is another common and preventable carcinoma worldwide; its annually rising incidence has made it a pre-eminent public health threat [4]. Malignant melanoma is a type of skin cancer and responsible for the vast majority of skin cancer deaths; it begins with the abnormal proliferation of cells known as melanocytes [5]. Chemotherapy, surgery, and radiotherapy are common cancer treatments, but their nonspecific action and severe side effects are cancer treatment's biggest obstacles [6,7]. To bypass the side effects, developing new green drugs, especially essential oils (EO) s, has recently received more attention [8,9]. Citrus aurantium (bitter orange) is a tree 4-6 m high, evergreen, hairless, with long blades of the Rutaceae (Citrus) family [10]. It possesses antispasmodic, antiinflammatory, anti-flatulence, antihypertensive, and diuretic properties [11][12][13]. Citrus limon (L.) or lemon is another member of the Rutaceae family rich in vitamins, minerals, dietary fiber, and carotenoids [14]. Citrus sinensis (scientifically name for the orange) grows as a fruit-bearing shrub with green leaves and white flowers and is also a member of the Rutaceae family [15,16]. EOs of the three mentioned specimens possesses anticancer effects against different types of breast cancer cell lines. Their IC 50 s against MCF-7 were reported at 82, 57, and 39 μg/mL, their IC 50 s against MDA-MB-231 were reported at 74, 37, and 39 μg/mL, and their IC 50 s against T47D were reported at > 300, 19, and 43 μg/mL [17]. Moreover, limonene (1-methyl-4-(1-methylethenyl) cyclohexane) is a major constituent in the mentioned EOs; it is a colorless and aromatic liquid oil and acts as a potential chemotherapeutic monocyclic monoterpene in nature [18][19][20]. Limonene has significant anticancer activity by inhibiting tumor initiation, growth and angiogenesis, and cancer cell apoptosis [21,22]. For instance, the antiproliferative activity of limonene on BW5147 cells, colon, gastro, melanoma, mammary gland tumors has been confirmed [23,24].
In this research, the anticancer effects of C. aurantium, C. limon, and C. sinensis EOs (CAEO, CLEO, and CSEO) and limonene were first investigated against A-375 (human melanoma cancer cell line) and MDA-MB-468 (human breast cancer cell line). By preparing chitosan nanoparticles (ChiNPs) containing them, an attempt was made to improve their anticancer properties.

Chemical composition of the EOs
Ingredients of CSEO, CLEO, CAEO were analyzed by Gas Chromatography-Mass Spectrometry (GC-MS); the EOs were analyzed using a 6890 GC system coupled with a 5975 series of mass selective detectors (Agilent Technologies, USA). The separations were performed on HP-5MS silica fused column (length, 30 m; internal diameter, 0.25 mm; film thicknesses, 0.25 mm; stationary phase, 5% phenyl 95% methyl polysiloxane). The column temperature program started at 40°C (fixed for 1 min), then raised with a rate of 3°C min − 1 to 250°C, and was finally held for 60 min at this temperature. The temperature of the injection port and detector was fixed at 250 and 230°C, respectively. Other operating conditions were as follows: carrier gas, helium (99.999%), split-flow 25 mL/min, septum purge 6 mL/min, and column flow rate 1 mL/min. Mass spectra were taken at full scan mode and 70 eV ionization energy and full scan mode. The scanned mass range was set at 50-550 m/z. The identification of ingredients was performed as described in our previous study, and the peak area normalization procedure was used for the quantitative determination of the compounds in the EOs [25].

Preparation of chitosan nanoparticles
Chitosan powder (0.25% w/v) was dissolved in a 1% acetic acid aqueous solution (4 h, 2000 rpm, ambient temperature). Preparation of chitosan nanoparticles containing limonene, CAEO, CLEO, and CSEO was obtained using a modified ionic gelation technique, as depicted in Fig. 1 [26]. Each EO or limonene (0.5% w/v) and tween 20 (0.5% w/v) was first mixed at 2000 rpm for 30 min at room temperature. The chitosan solution was then added and stirred for another 30 min. After that, an aqueous solution of TPP (0.15% w/v) was added 1 mL/h using the syringe pump, and the mixture was stirred for 40 min (2000 rpm) to stabilizing the nanoparticles. The prepared samples were abbreviated as CLChiNPs, CSChiNPs, CAChiNPs, and LimChiNPs. Moreover, the same methodology was used to prepare free chitosan nanoparticles (ChiNPs), but no EO or limonene was used.

Characterization of the prepared nanoformulation Size analysis
Dynamic light scattering (DLS) technique was used to analyze all nanoformulations particle sizes (dynamic light scattering, scatter scope, K-ONE NANO. LTD, Korea). D50 was considered particle size, and particle size distribution (SPAN) was calculated as d90-d10/d50. D is diameter, and 10, 50, and 90 are percentile of particles with a smaller diameter than these specified diameters. Formulations with SPAN values less than 1 possess narrow particle size distributions [27].

Investigation of the anticancer activity
The anticancer activity of limonene, CAEO, CLEO, and CSEO (as non-formulated samples) also, Lim-ChiNPs, CAChiNPs, CLChiNPs, and CSChiNPs (as nanoformulations) were investigated using MTT assay. The EOs and limonene were dissolved (0.5% w/v) in a PBS solution containing 0.5% DMSO. The cell lines were cultured in 75 cm 2 culture flasks in DMEM medium supplemented with 10% of FBS and 1% of penicillin-streptomycin (P/S) and incubated at 37°C air (95%) and CO 2 (5%). Cells (MDA-MB-468 and A-375) were separated by trypsin; they were then seeded (1 × 10 4 cells per well) in 96 well plates and incubated overnight for attachment. The following day, the culture media was discarded, and a 75 μL complete fresh medium was added to each well. By adding an appropriate amount of the samples, concentrations were finally fixed at 1200, 600, 300, 150, and 75 μg/mL. The treated plates were incubated for 24 h at 37°C with CO 2 5%, and their content was then discarded, and wells were washed with 100 μL PBS to remove nanoformulations milky color. After that, 100 μL MTT reagent (0.5 mg.mL − 1 ) was added to each well and were incubated for another 4 h; created formazan crystals were then dissolved by adding DMSO (100 μL/well). The control group (six-well in each plate) was treated only with 25 μL PBS (0.5% DMSO) and 75 μL culture media. Finally, the absorbance (A) of the wells was monitored by an ELISA plate reader at 570 nm. The cell viability at each concentration was calculated using the following equation:
The particle size of the prepared chitosan nanoparticles containing limonene or EOs DLS analyses of the prepared nanoformulations are given in Fig. 2

Successful loading of limonene or EOs in chitosan nanoparticles
The ATR-FTIR spectrum of ChiNPs is depicted in Fig. 3A; the bond at about 1700 cm-1 can be related to carbonyl stretching of the pure chitosan's secondary amide band and carbonyl group tween. The characteristic peak at 1094 cm − 1 relates to symmetric and antisymmetric stretching vibrations in the PO 2 group, and the strong band at 1020 cm − 1 belongs to symmetric and anti-symmetric stretching vibrations in the PO 3 group. After the crosslinking process, two bands at 1280 and 1152 cm − 1 belong to anti-symmetric stretching vibrations of PO 2 groups in TPP ions. These new peaks confirmed ionic crosslinks between protonated amino groups of chitosan and tripolyphosphate anionic groups. In the ATR-FTIR spectrum of limonene (Fig. 3B), the band at 3001 cm − 1 relates to = C-H, and the bands at 2962, 2913, 2855, and 2855 cm − 1 showed -CH stretching, the specific absorption at 1677 cm − 1 corresponds to the stretching vibration of vinyl substituted C=C. The absorption band at 1644 cm − 1 is assigned to the C=C vibration. The characteristic peaks at 989 and 890 cm − 1 attributed to C-H bending absorption.
In the spectrum of LimChiNPs (Fig. 3C), the characteristic peak at 2925 m − 1 is showed C-H stretching in chitosan and C-H of limonene. A strong characteristic peak showed at 1278 cm − 1 belong to C-N stretching indicates the complex formation via electrostatic interaction between NH3 + groups of chitosan within phosphoric groups of TPP. The band around 1580 cm − 1 can be assigned to C-N stretching vibration and refers to the amide group because of the NH 2 bending vibration. The band at 1017 cm − 1 related to C-O in chitosan and the shape and position of the peaks proved that limonene was successfully encapsulated ChiNPs.
ATR-FTIR spectrum of CAEO is depicted in Fig. 3D; the broad peak at 3436 cm − 1 attributes to NH, the bands at 3761 related to = C-H. The bands at 2961, 2923, 2872 cm − 1 displayed -CH cm − 1 , and the peak at 1706 cm − 1 corresponding to the stretching vibration of carbonyl C= O. Absorption bands at 1644 and 1437 cm-1 are assigned to the aromatic ring C=C skeleton vibration of an aromatic substance. The peak at 1022 cm − 1 is characteristic of a stretching vibration of C-N. The peak at 957 cm − 1 is attributed to C-H bending absorption, and the strong peak at 758 cm − 1 is assigned to benzene rings C-H vibration absorption.
From Fig. 3E, some of the specific peaks of CAEO disappeared when it was encapsulated in chitosan, i.e., CAChiNPs. A strong new peak showed at 1281 cm − 1 belong to the C-N stretch indicating the complex formation via electrostatic interaction between NH3 + groups of chitosan within phosphoric groups of TPP. Also, the band around 1547 cm − 1 can be assigned to C-N stretching vibration and refers to the amide group because of the NH 2 bending vibration.
In ATR-FTIR spectra of CLEO, Fig. 3F, the bands at 3399 cm − 1 related to OH stretching vibration, the bands at 3072 cm − 1 related to = C-H. The bands at 2963, 2918, 2855, and 2834 cm − 1 showed -CH stretching vibration, In ATR-FTIR of CLChiNPs (Fig. 3G), the new strong peak at around 1280 cm − 1 relates to C-N stretching was attributed to the electrostatic interaction between chitosan and TPP. Also, the sharp peak around 1545 cm − 1 attributed to C-N stretching vibration related to the amide group because of the NH 2 bending vibration. All other characteristic peaks appear in the spectra of CLEO and ChiNPs; it is confirmed that CLEO was successfully encapsulated in ChNPs.
In the spectrum of CSEO (Fig. 3H), the bond at about 3412 cm − 1 can be related to OH stretching vibrations, and the peaks at around 2964, 2918, 2885 cm − 1 are related to -C-H. The vibrational bands around 1729 cm − 1 related to C=O, and the band around 1677 attribute C-O stretching vibrations. The main peaks around 1110, 1115, and 1309 cm − 1 are related to C-O. The peaks at 885 cm − 1 attributed to C-H bending absorption.
After CSEO was encapsulated, the spectrum showed a strong peak at 1280 cm − 1 belongs to the C-N stretch, indicating the complex formation via electrostatic interaction between chitosan and TPP (see Fig. 3I). The   Table 2.
Interestingly, the viability of A-375 cells treated with CLChiNPs, CSEO, and CSChiNPs at all examined concentrations (150, 300, 600, and 1200 μg/mL) was reduced < 20%. IC 50 s of CLChiNPs, CSEO, and CSChiNPs were 0.124, 0.02, and 0.03 μg/mL. Besides, IC 50 s of LimChiNPs and CAChiNPs were observed at 30.24 and 55.00; there was no significant difference together (p > 0.05). However, their potency was significantly more than their no-formulated forms, i.e., limonene and CAEO, with IC 50 s of 246.05 and 10,564.00 μg/mL. From Fig. 5, the cytotoxicity effects of limonene, CAEO, CLEO, and CSEO on MDA-MB-468 cells are depicted. Generally, the samples had less potency on the growth of this cell line than A-375 cells; viability of cells reduced to lower than 10% after treating with only Lim-ChiNPs at a concentration of 1200 μg/mL. The bestobtained IC 50 s were related to CSChiNPs (23.65 μg/mL) and CLChiNPs (40.32 μg/mL). Moreover, IC 50 s of Lim-ChiNPs and CAChiNPs (650.70 and 240.44 μg/mL) were significantly more potent than their non-formulated forms (2118.94 and 2037.53 μg/mL).
Interestingly, no report was found on preparing chitosan nanoparticles containing CAEO, CLEO, CSEO, and limonene as anticancer agents on these cell lines in the literature. However, some reports on the preparation of some formulations have been found. For instance,  chitosan microcapsules containing limonene with a particle size of 2-12 μm were reported [33]. Electrosprayed Alyssum homolocarpum seed gum nanoparticles containing limonene with a mean diameter of 65.68 ± 8.80 nm was reported in another research [34]. In a study, CAChiNPs with a particle size of 40 nm was proposed as mushroom food packaging; it significantly decelerated the rate of color change, weight loss, and firmness compared to fumigation with EO [35]. In another study, by incorporating CAEO in ChiNPs (20-60 nm), antioxidant and antimicrobial effects were improved [36]. Chitosan nanoemulsion containing CSEO as a green fruit juice preservative was also reported [37]. In another report, CLEO was encapsulated in chitosan/hicap with a mean particle size of 339.3 nm; it was concluded that due to the desirable physicochemical properties and thermal stability, this formulation could be used in medicine and food industries [38]. Furthermore, the preparation of nanoformulations to improve the anticancer effects of EOs has recently received much attention [39]. For instance, a study confirmed that by loading limonene into ChiNPs with a particle size of 339.5 nm, its antioxidant became more potent by improving its solubility [40]. CAEO nanoemulsion showed cytotoxicity against A549 cells with the IC 50 value of 152 μg/mL [41]. Besides, isolated nanovesicles from C. lemon have been shown an inhibitory effect in a time-dependent manner on cancer cell proliferation in different tumor cell lines by inducing apoptotic cell processes [42]. It has also been proposed that loading limonene into solid lipid nanoparticles (one of the pharmaceutical drug delivery systems) can reduce growth percentages in cancer cells with a low toxic effect on the non-tumoral cell line [43]. In vitro cytotoxicity assay showed that D-limonene-loaded niosome had a noticeable anticancer effect than D-limonene against HepG2, MCF-7, and A549 cell lines [39].
In the current study, by preparing ChiNPs containing EOs/limonene, their anticancer effects were improved, e.g., IC 50 s of limonene against A-375 and MDA-MB-468 were obtained at 246 and 2118 μg/mL, while IC 50 s of its nanoformulated state (LimChiNPs) were observed at 30 and 650 μg/mL. CAEO did not show proper efficacy on both mentioned cell lines (IC 50 10,564 and 2037 μg/mL). However, after the preparation of ChiNPs containing CAEO, its IC 50 s were reduced dramatically to 55 and 240 μg/mL. Moreover, only IC 50 of CSEO (0.02 μg/mL) against A-375 cells was not improved after preparation of its nanoformulation (CSChiNPs: 0.03 μg/mL); further investigation is needed at low concentrations. However, as the hydrophobic nature of CSEO, for its practical administration in vivo or clinical trials, it should be formulated; thus, its nanoformulated form (CSChiNPs) was the best sample against A-375 cells. Interestingly, IC 50 s of CSChiNPs and CLChiNPs (0.03 and 0.124 μg/mL) against A-375 were more potent than cisplatin with IC 50 of 0.40-0.45 μg/mL [29,44]. Their efficacies against MDA-MB-468 (23.65 and 40.32 μg/mL) were also comparable or more potent than cisplatin (IC 50 32.50 μg/ mL) [45].

Conclusions
Compounds of three EOs from the Citrus family, including C. aurantium, C. limon, and C. sinensis, were first identified using GC-MS analysis. Their anticancer effects were then investigated on human breast cancer and melanoma cell lines. After that, chitosan nanoparticles containing EOs and limonene (as their major ingredients)