It has been shown that the formation of hydrophobic drug-loaded nanomedicines not only improves their stability, solubility, bioavailability, biodistribution, pharmacokinetics, and pharmacodynamics, but also brings about a decline in the toxicity of the drug [29, 30]. The obtained results in terms of the extract composition, formulation characterization, and the anticancer effect of the obtained nanoparticles are discussed below.

After analyzing the RDEO extracts by GC-MS, 39 major compounds with their respective peak areas were found. By comparing the mass fragmentation patterns of comparable compounds obtained for the extract with the data available in the WILEY library, different compounds were identified and listed in Table 2. The most important compounds were beta-citronellol (31.91%), nonadecane (21.43%), heneicosane (11.80%), geraniol (9.86%), 9-nonadecene (4.17%), eicosane (2.76%), heptadecane (2.27%), tricosane (2.12%), germacrene-D (1.81%), methyleugenol (1.65%), nerol (1.16%), linalool (0.83%), alpha.-guaiene (0.68%), 1,8-cineole (0.64%), alpha-humulene (0.57%), cis-farnesol (0.53%), 4-terpineol (0.52%), aciphyllene (0.52%), trans-caryophyllene (0.37%), geranyl acetate (0.36%), cis-2,6-dimethyl-2,6-octadiene (0.34%), octadecane (0.34%), (-)-alpha-terpineol (0.29%), rose oxide trans (0.20%), heneicosane (0.20%), Y-gurjunene (0.19%), 8-heptadecene (0.19%), (9Z)-tricosene (0.19%), dl-limonene (0.17%), beta-bourbonene (0.16%), 3-eicosene, (E)-(0.16%), 1-nonadecene (0.15%), eugenol (0.14%), alloaromadendrene (0.14%), pentadecane (0.12%), 1-nonadecene (0.12%), alphPinene (0.11%), 10-heneicosene (c,t) (0.11%), and rose oxide (0.08%). The results showed that 31.91% of R. damascena consisted of beta-citronellol. Chemical compositions of R. damascena reported from different regions of the world entail citronellol, geraniol, nerol, phenylethyl alcohol, nonadecane, nonadecene, eicosane, heneicosane, tricosane, alpha-guaiene, geranyl acetate, and eugenol [6].

Citronellol is a monoterpenoid with the molecular formula of C10H20O. This chemical can prevent the activities of Staphylococcus aureus and Salmonella typhi. It also possesses a potent inhibitory effect on Candida albicans. Treatment with citronellol in patients receiving radiotherapy and/or chemotherapy has been demonstrated to mitigate the adverse effects of therapy (e.g., dysgeusia, nausea, hearing loss, and numbness of the extremities) and lessens the depletion of leukocytes and neutrophils to ameliorate their immune function [31]. Roses have been shown to have effective healing properties owing to their abundance of beneficial components, fragrant compounds (EOs such as monoterpenes and sesquiterpenes), hydrolysable and condensed tannins, and secondary metabolites such as flavonoids (flavonols, flavones, and anthocyanins). Rose EOs and extracts have therapeutic attributes, including respiratory antiseptics, anti-inflammatories, antioxidants, expectorants, mucolytics, and decongestants. They also can act as symptomatic prophylactics and drugs, thereby alleviating dramatic suffering during severe diseases [32].

Data and research on the delivery of RDEO using NLCs in the literature are very limited. In this study, RDEO-NLC using the ultrasonication method is reported. The lipid matrix of NLC contains a mixture of solid and liquid lipids (oil), which decreases the melting point of the solid lipid [33]. GMS and SA were employed as solid lipids and OA (liquid oil). In the development of NLC, surfactants reduce the interfacial tension between the lipid and the aqueous phase and, therefore, contribute to the stability of the resultant formulation [33]. In nanoparticles, the selection of a surfactant mixture is performed considering the hydrophilic-lipophilic balance (HLB) of the lipids constituting the nanoparticle matrix and their concentration in the lipid phase of the dispersion [33]. Tn80, Sn60, and Sn80 were the three surfactants selected in this study. Stable nanoemulsions are formed when the aqueous phase, oil phase, HLB, and surfactant concentration are fully matched in the right sequences [34]. As represented in Table 1, four formulations with different amounts of starting materials were prepared to optimize the formulation. Among the four formulations, RDEO-NLC2 was selected as the optimized formulation due to having a smaller particle size with an acceptable PDI and zeta potential. The optimal formulation (RDEO-NLC2) consists of 0.1 g RDEO and OA (liquid lipid, 0.03 g), as well as Sn80 (0.1 g) and Tn80 (0.2 g) as surfactants. The average particle size of the optimal formulation was 78.39 ± 1.52 nm, and its polydispersity index (PDI) and ZP were 0.28 ± 0.01 and − 31.0 mV, respectively (Table 1). The RDEO-NLC2 exhibited an encapsulation efficiency of 99.6% and a loading capacity of 9.6%, with a higher than ZP (± 30 mV), which indicates that the obtained nanoparticle should be stable [35] (Fig. 1). 

Zeta potential (A) and particle size (B) distribution of optimized NLC formulation (RDEO-NLC2)

TEM is one of the most suitable tools for evaluating and analyzing the morphology of nanostructured particles. The morphology of the optimized formulation (RDEO-NLC2) was determined and depicted in Fig. 2A. The results demonstrated a relatively spherical shape for RDEO-NLC2. The relevant histograms (frequency v size) attained by statistical analysis of ~ 500 particle size distributions of RDEO-NLC2 particles were observed to be smaller than 270 nm with a mean particle size of 81.98 ± 49.64 nm (Fig. 2B).

TEM image (A) and particle size (B) distribution histogram of optimized NLC formulation (RDEO-NLC2)

The MTT method was utilized to check the cell viability and the toxicity effect of cisplatin, RDEO, placebo, and NLC-loaded with RDEO-NLC2 on the MDA-MB-231 BC cell line at 24 and 48 h. The MDA-MB-231 cells were incubated with RDEO, RDEO-NLC2, and placebo at 6.25, 12.5, 25, 50, and 100 ppb concentrations and also with cisplatin in 1, 5, 10, 20, and 50 μg/ml concentrations at 37 °C for 24 and 48 h (Fig. 3). In this study, the conventional chemotherapy drug cisplatin, which contains platinum, was used as a model anticancer drug to be compared to RDEO-NLC2 in terms of cell viability. Cisplatin has undesirable side effects, including allergic reactions, drug resistance, severe kidney problems, decreased immunity to infections, gastrointestinal disorders, hemorrhage, and hearing loss, especially in younger patients [36]. Considering the common side effects of cisplatin, NLC-RDEO2 was formulated to reduce these side effects and to replace cisplatin. In the present study, untreated cells were considered as control. The results presented in Fig. 3 revealed that the RDEO-treated MDA-MB-231 cells did not indicate any toxicity and significant adverse effects on cell proliferation, even at a 100 ppb concentration at 24 and 48 h. After treating the cells with RDEO-NLC2 and cisplatin, cell toxicity was dramatically increased. Based on the MTT assay, cisplatin, followed by RDEO-NLC2, was explored to be more cytotoxic to MDA-MB-231 BC cells after 48 h (Fig. 3). The results also showed that the effect of both cisplatin and RDEO-NLC2 on the MDA-MB-231 cells was dose-dependent (p < 0.001). By increasing the dose of cisplatin to 50 μg/ml and RDEO-NLC2 to 100 ppb, significant effects were found on cell proliferation, and cell survival decreased at 24 and 48 h. The cell viability, however, declined to 38.88 ± 8.63% and 25.33 ± 1.4% (p < 0.001 for both) after exposure to 50 mg/ml of cisplatin after 24 h and 48 h, respectively (Fig. 3). Also, the cell treatment with RDEO-NLC2 complex significantly lessened the viability of the cell to 52.74 ± 1.34% and 39.23 ± 4.92% (p < 0.001 for both) in 100 ppb concentration after 24 h and 48 h, respectively. Based on the results obtained, the cisplatin drug had more negative effects on cell proliferation and higher toxicity than RDEO-NLC2 and placebo at 24 and 48 h. According to the present study and previously published data, nanostructures containing plant EOs with cytotoxic effects are very favorable in drug delivery and cancer control and treatment. Advances in this field reduce the many side effects of cancer treatment. In a previous survey, Kryeziu et al. concluded that nanoencapsulation of Origanum vulgare L. EO into liposomes could contribute to the preservation and improvement of its antioxidant and cytotoxic activity, as well as produce nanocarriers for the development of anticancer agents [37].

Cell viability of MDA-MB-231 cells incubated with different concentrations (100, 50, 25, 12.5, and 6.25 ppb) of RDEO, placebo, and RDEO-NLC2 for 24 h (A) and 48 h (C) and concentration (50, 20, 10, 5, and 1 μg/ml) of cisplatin for 24 h (B) and 48 h (D)

In a separate investigation, researchers explored the potential of nanoemulsions containing Mentha piperita essential oil (MPEO) [38]. This essential oil’s anticancer properties were assessed using three distinct human breast cancer cell lines, namely, MCF-7, MDA-MB-231, and MDA-MB-468. The study sought to determine the anticancer effects of MPEO on all of these subtypes of human breast cancer cell lines. Remarkably, the study revealed that the anticancer impact of MPEO achieved within a 24-h treatment using the newly developed nanoemulsions was significantly better than the essential oil with an exposure time of 72 h. The nanoemulsions exhibited excellent cytotoxicity against all the cell lines across all incubation timeframes. These findings hold promise for the potential use of MPEO-loaded nanoemulsions as a novel and effective approach for combating various subtypes of human breast cancer cells [38].

After incubating the cell line MDA-MB-231 with the IC50 concentration of RDEO-NLC2, cisplatin, and placebo, compared to the control group, at 24 and 48 h, the value of both apoptotic and necrotic cells was determined by flow cytometry. As illustrated in Fig. 4, the percentages of viable (Q4), early (Q3) and late (Q2) apoptotic, and necrotic (Q1) cells in treated cells were different from those in untreated cells. The percentage of viable cells suggested a significant (p < 0.001) decline in the cell viability from 90.53 ± 0.23 and 88.43 ± 2.61 in control cells to 75.00 ± 1.63 and 66.83 ± 1.96 in RDEO-NLC2-treated cells and 62.13 ± 5.08 and 57.03 ± 2.57 in cisplatin-treated cells at 24 and 48 h respectively. According to the results, most of the MDA-MB-231 cells treated with cisplatin and RDEO-NLC2 in the early apoptotic stage at 24 h could remarkably prevent the proliferation of the cancer cells, while at 48 h, the effect of RDEO-NLC2 on these cells was greater than cisplatin. Moreover, cancer cells have evolved complicated mechanisms to antagonize necroptosis and apoptosis. Hence, triggering a single type of programmed cell death may be insufficient for treating cancer metastasis. The selection of varying inducers of cell death or the combined use of various pathway inducers of cell death can help overcome drug resistance to eliminate metastatic cells [39] (Fig. 5).

Apoptosis assay of MDA-MB-231 RDEO-NLC2 IC50-treated cells (A), cisplatin IC50-treated cells (B), placebo IC50-treated cells (C), and control cells (D) for 24 h. The quantitative analysis was plotted to show the population of VC (viable cells), EA (early apoptotic cells), LA (late apoptotic cells), and NC (necrotic cells) cells in 24 h (E)

Apoptosis assay of MDA-MB-231 RDEO-NLC2 IC50-treated cells (A), cisplatin IC50-treated cells (B), placebo IC50-treated cells (C), and control cells (D) for 48 h. The quantitative analysis was plotted to show the population of VC (viable cells), EA (early apoptotic cells), LA (late apoptotic cells), and NC (necrotic cells) cells in 48 h (E)

There is a limited number of studies exploring the impact of Rosa damascena essential oil–loaded nanostructured lipid carriers on breast cancer. However, it is interesting to note a relevant study that focused on the effects of Rosa damascena Mill on colorectal cancer [40]. In this particular research, the findings indicated that R. damascena callus, induced by L-ascorbic acid, exhibited the potential to enhance both growth and secondary metabolite contents. Moreover, it demonstrated significant anti-proliferative, anti-clonogenic, and anti-migratory effects on Caco-2 cancer cells, suggesting its potential as an adjunctive therapy in the context of cancer treatment. This study sheds light on the broader applications of Rosa damascena in the realm of cancer research, potentially extending to breast cancer as well. Further exploration of its efficacy in treating breast cancer through nanostructured lipid carriers remains a promising area for future investigation.