Enhanced cytotoxic activity of Moringa oleifera-loaded pharmacosomes against neuroblastoma

Abstract

Moringa oleifera is known for its diverse therapeutic properties, including anticancer effects attributed to its rich phytochemical profile; yet, its poor solubility and stability limit its therapeutic translation. In this study, phospholipid-based pharmacosomal nanocarriers loaded with Moringa oleifera extract (MO) were developed and characterized to enhance its delivery and anticancer efficacy against neuroblastoma. Neuroblastoma is a common pediatric malignancy of neural crest origin with poor prognosis in high-risk cases, highlighting the need for safer and more effective therapeutic strategies. GC-MS analysis of the MO extract revealed diverse bioactive constituents with documented cytotoxic, pro-apoptotic, and antioxidant properties, including polyphenolic acids, flavonoid derivatives, nootkatone, uridine, and fatty acid derivatives. MO-loaded pharmacosomes (MO-PhS) formulated using lecithin and chitosan exhibited favorable physicochemical characteristics, including nanoscale size (41.07–190.12 nm), high entrapment efficiency (94.52 ± 2.79%), spherical morphology (PDI 0.29 ± 0.04), strong negative surface charge (−58.73 ± 0.99 mV), and enhanced thermal stability. In vitro cytotoxicity studies on SH-SY5Y neuroblastoma cells demonstrated a marked enhancement in anticancer activity for MO-PhS, achieving approximately a 10-fold reduction in IC₅₀ compared to free MO. Cell cycle analysis by flow cytometry revealed a pronounced redistribution of treated cells from G0/G1 into S and G2/M phases, indicating disruption of proliferative progression and replication stress, with pharmacosomal delivery of MO producing a more pronounced S-phase accumulation. Notably, these cell cycle perturbations were achieved at an approximately 10-fold lower concentration for MO-PhS compared to the free extract, underscoring the superior dose-efficiency of pharmacosomal delivery. Plain pharmacosomes exhibited minimal cytotoxicity against human normal fibroblasts (hFB), confirming carrier biocompatibility. These findings highlight MO-PhS as a promising nanoplatform for neuroblastoma therapy, offering enhanced cytotoxic efficacy, higher stability and antioxidant properties, and excellent biocompatibility, warranting further preclinical investigation.

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1. Introduction

Neuroblastoma is the most common extracranial solid tumor occurring in childhood and accounts for 15% of pediatric cancer deaths.^1,2 Despite advances in chemotherapy, radiotherapy, and surgical interventions, high-risk neuroblastoma remains associated with poor long-term survival and significant treatment-related toxicity.² Thus, there is a critical need for novel, safe, and more effective therapeutic strategies that target tumor cells while minimizing systemic side effects.

Natural products derived from medicinal plants have gained increasing attention for cancer treatment, offering diverse bioactive compounds with antioxidant, anti-inflammatory, and cytotoxic activities.^3,4 Commonly recognized as the “tree of life” or “miracle tree”, Moringa oleifera is widely utilized as a functional food and nutritional supplement around the world.^5–8 M. oleifera has been extensively used in traditional and ethnomedicine, which suggests that it may have multifaceted preventive and therapeutic activities against a wide range of human disorders.^5–8 In fact, M. oleifera, particularly leaves, has been positively correlated with a number of pharmacological benefits, showing outstanding therapeutic activities such as antihypertensive, antispasmodic, anticoagulant, antiviral, antidiabetic, antiulcer, antipyretic, anti-inflammatory, antimicrobial, anticancer, hepatoprotective, immunostimulant, and cardiotonic effects.^5–15

Moreover, M. oleifera compromises plenty of vitamins, oils, proteins, minerals, lipids, metal ions, and phenolic ingredients. Phenolic compounds entailed are mostly carotenoids, flavonoids, and phenolic acids, which are effective against malnutrition and other illnesses.^16–23 Significantly, the United Nations has recommended using M. oleifera as a nutritional supplement to the human diet, supporting the high safety profile of this plant.²⁴ Also, a daily intake of 1 g Kg⁻¹ of M. oleifera has been reported to be safe for human consumption.²⁵ Recent experimental investigations reported neuroprotective effects of M. oleifera against several neurodegenerative disorders, including dementia,²⁶ Alzheimer's disease,^27,28 PD,^29–31 and stroke.^32,33

Furthermore, M. oleifera has been shown to contain several phytochemicals such as flavonoids, phenolics, sterols, fatty acids, and terpenoids, many of which have shown cytotoxicity against various cancer cell lines, including breast, colon, and liver cancers.^34–36 Also, the cytotoxic effects of M. oleifera have been explained by several mechanisms exerted by its extracts, including significant suppression in Nrf2 mRNA level and a corresponding decrease in protein expression, intracellular induction of ROS and reduction of glutathione levels, and promotion of pro-apoptotic action (via increase in p53 protein expression, caspase-9, caspase-3/7 activities, etc.) in tumor cells.^34–36 These mechanisms strongly support the role of M. oleifera in antiproliferation and potent induction of apoptosis in cancer cells.

Nevertheless, the main challenges facing the clinical applications of natural extracts, including M. oleifera, generally include poor aqueous solubility, low bioavailability, and limited stability in physiological environments.^12,19,37,38 To overcome these limitations, the use of nanocarrier systems has emerged as a promising approach to enhance the delivery and efficacy of plant-derived compounds.

Pharmacosomes, a class of lipid-based nanocarriers composed of phospholipid complexes (typically lecithin) and stabilizing agents such as chitosan, offer several advantages for drug delivery, including enhanced solubility, stability, site-specific targeting, and controlled release.^39–42 They share similar structural characteristics with lipid-based nanocarriers, particularly the presence of a bilayer membrane, allowing them to effectively encapsulate both hydrophilic and lipophilic agents. This dual loading capacity enhances the solubility, stability, and bioavailability of poorly water-soluble natural extracts, while also improving controlled release and site-specific targeting.^39,40

In comparison to conventional liposomes, pharmacosomes offer several advantages, including superior physicochemical stability, less susceptibility to oxidative degradation, and improved drug entrapment efficiency.^39,40,43 Moreover, their reduced likelihood of drug leakage during storage or circulation contributes to better shelf life and therapeutic performance, features particularly important for natural products such as M. oleifera extracts, which often exhibit poor pharmacokinetic profiles and instability under physiological conditions.⁴⁴ Additionally, pharmacosomes do not require cholesterol for bilayer stabilization, which minimizes formulation complexity and potential interference with bioactivity. Their self-assembling ability and relatively simple preparation methods further support their scalability and industrial feasibility.^39,40

In addition to improved stability and delivery efficiency, pharmacosomes are reported to enhance the cytotoxic efficacy of loaded bioactives by promoting stronger membrane interaction, improved intracellular accumulation, and sustained exposure of tumor cells to therapeutic compounds. Such features have translated into greater antiproliferative and pro-apoptotic activity compared to free drugs or conventional lipid carriers in several cancer models.³⁹ Also, pharmacosomes exhibit enhanced targeting efficiency, making them particularly suitable for treating and targeting various malignancies including neuroblastoma. Their nanoscale size and optimized surface characteristics can facilitate enhanced permeability and retention (EPR) in tumor tissues and potentially support targeted delivery for treating cancers such as neuroblastoma.⁴⁵ Recent studies have demonstrated that pharmacosome formulations significantly increase BBB penetration and brain accumulation of therapeutic agents, supporting their application in neurological disorders.⁴⁵

In this study, pharmacosome nanoparticles were prepared as a delivery system for M. oleifera extract. Their physicochemical properties, thermal stability, antioxidant capacity, and cytotoxic activity against SH-SY5Y neuroblastoma cells were investigated, aiming to explore their potential as a natural-product-based anticancer platform for treating neuroblastoma.

2. Materials and cell culture

2.1. Materials

Fresh leaves of M. oleifera were purchased from a local vendor (Cairo, Egypt). Lecithin was purchased from VWR BDH Chemicals (Fontenay-sous-Bois, France). Low molecular weight chitosan was supplied by CDH Fine Chemicals (New Delhi, India). Phosphate buffer saline (PBS) tablets were supplied by Genetix (Genetix Biotech, Asia, Pvt., Ltd). Chloroform was purchased from Chem-Lab NV, Belgium. DPPH was provided by Sigma (Sigma-Aldrich Co., Germany). DMSO was obtained from Fisher Scientific (UK). KBr (FTIR grade) was purchased from Merck (KGaA, Darmstadt, Germany). MTT was provided by SERVA GmbH (USA). Dimethyl sulfoxide (DMSO) was obtained from Merck (Darmstadt, Germany).

2.2. Cell culture

SH-SY5Y cells, isolated from a bone marrow biopsy taken from a four-year-old female with neuroblastoma, were purchased from ATCC, USA. The cells were routinely cultured in high-glucose DMEM. It was supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and contained 100 units per mL of penicillin G sodium and 100 units per mL of streptomycin sulphate. All from PAN-Biotech, (Germany). Cells were maintained at sub-confluency at 37 °C in humidified air containing 5% CO₂. For sub-culturing, monolayer cells were harvested after trypsin/EDTA treatment at 37 °C. Cells were used when confluence had reached 75%.⁴⁶

3. Methods

3.1. Preparation and Soxhlet extraction of Moringa oleifera leaf extract (MO)

Fresh M. oleifera leaves were thoroughly washed with tap water to remove surface impurities and subsequently air-dried in a dark, well-ventilated environment at 20 °C to preserve their bioactive compounds. Once dried, the leaves were finely ground into a uniform powder using a high-speed blender and stored under refrigeration for further use. For extraction, the Soxhlet method was employed, utilizing a 90% (v/v) methanolic solution as the solvent. A leaf powder-to-solvent ratio of 1 g per 10 mL of methanol (90%) was maintained, and the extraction process was carried out at a controlled temperature range of 60–70 °C. The extraction continued for approximately 9 h until the solvent exhibited a pale greenish coloration, indicating the depletion of soluble compounds. The obtained M. oleifera extract (MO) was subsequently collected, dried in a desiccator at ambient temperature, and stored at 4 °C to retain its stability and bioactivity.⁴⁷

3.2. Preparation of pharmacosome nanostructures

Three pharmacosome formulations (PhS1, PhS2, and PhS3) were prepared using the thin-film hydration method, with some modifications.⁴⁵ In each formulation, lecithin and low molecular weight chitosan were dissolved in 5 mL of tetrahydrofuran (THF) inside a sealed glass container. The mixture was then transferred into a 100 mL round-bottom flask and subjected to rotary evaporation (45 min) until a thin lipid film was formed on the flask walls. The lipid film was then hydrated using phosphate-buffered saline (PBS, pH 7.4) under rotary evaporation for at least 1 h to ensure complete hydration. The final pharmacosome dispersions were stored at 4 °C for further characterization. PhS1 and PhS2 varied in lecithin concentration, whereas PhS3 incorporated 0.4% (w/v) of MO extract into the formulation (Table 1).

Table 1 Composition of pharmacosome formulations. (The concentration of Moringa extract in PhS3 was selected based on initial screening studies conducted on neuroblastoma cells)^a

Formulation	Lecithin (% w/v)	Chitosan (% w/v)	MO (% w/v)	Solvent (THF)	Hydration medium (PBS, pH 7.4)
a Abbreviations: THF = tetrahydrofuran; PBS = phosphate buffer saline; PhS = pharmacosomes; MO = Moringa oleifera leaf extract.
PhS1	4.5%	0.25%	—	5 mL	5 mL
PhS2	2.25%	0.25%	—	5 mL	5 mL
PhS3	2.25%	0.25%	0.4%	5 mL	5 mL

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3.3. Characterization tests

3.3.1. GC-MS analysis. The MO extract was derivatized with bis(trimethylsilyl)trifluoroacetamide (300 µL, 80 °C, 2 h) to volatilize polar compounds. Chromatographic separation was performed on an Agilent 7890B GC/5977A MSD system equipped with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 µm). Helium carrier gas was maintained at 8.2 psi (constant pressure mode). The oven program was held at 60 °C for 5 min, ramped at 20 °C min⁻¹ to 300 °C, and held for 5 min. Injection was performed in split-less mode (1 µL, 300 °C). Mass spectrometry operated in electron ionization (EI) mode (70 eV), scanning m/z 50–550 with an 8.0 min solvent delay (ion source: 230 °C; quadrupole: 150 °C). Compounds were identified using the NIST/EPA/NIH mass spectral library (v2.2, 2014) with ≥80% similarity matching.^48,49

3.3.2. Fourier transform infrared (FTIR) spectroscopic characterization. FTIR spectroscopy was employed to chemically characterize the MO extract, plain pharmacosomes (PhS), and MO-loaded pharmacosomes (MO-PhS). Analysis was conducted using a Nicolet 380 FTIR (Thermo-Scientific, Madison, USA), applying the potassium bromide (KBr) disc method. Spectral data were recorded within the frequency range of 4000–400 cm⁻¹ at a resolution of 4 cm⁻¹.

3.3.3. Thermogravimetric analysis (TGA). The thermal stability and degradation behavior of the MO extract and both plain and loaded pharmacosomal nanoformulations were assessed using a TGA Q50 thermogravimetric analyzer (TA Instruments, New Castle, DE, USA). Accurately weighed samples were subjected to a controlled heating process from 25 °C to 600 °C at a constant rate of 10 °C min⁻¹ under a nitrogen atmosphere (flow rate: 50 mL min⁻¹). The corresponding weight loss of each sample was continuously recorded as a function of temperature, providing insights into their thermal decomposition profiles.

3.3.4. Nanoparticle morphology and encapsulation analysis by UHR-TEM. The encapsulation efficiency of MO extract within MO-PhS and their morphological characteristics were examined using ultra-high resolution transmission electron microscopy (UHR-TEM) (JEOL JEM-2100 Plus, Tokyo, Japan) operated at 200 kV, following established protocols for lipid-based nanoparticles. Briefly, nanoparticle suspensions were prepared in distilled water, sonicated (37 °C, 10 min), and deposited onto 200-mesh copper grids coated with a 1 nm carbon film. Samples were negatively stained with 2% (w/v) uranyl acetate for 30 s, blotted dry with filter paper, and air-dried prior to imaging. Morphological analysis was performed at magnifications ranging from 5000× to 1 000 00× to assess nanoparticle size, uniformity, and encapsulation integrity.

3.3.5. Dynamic light scattering (DLS) and zeta potential analysis. The hydrodynamic diameter (Z-average), polydispersity index (PDI), and zeta potential of both PhS and MO-PhS were characterized using a Nano-Zetasizer system (Malvern Instruments Ltd, UK). For DLS measurements, samples were diluted in phosphate-buffered saline (PBS) (1 : 10, w/v) and analyzed at 25 °C. Zeta potential was measured in the same instrument using laser Doppler electrophoresis, with samples appropriately dispersed in PBS to maintain consistent ionic strength. All measurements were performed in triplicate.

3.3.6. Encapsulation efficiency (EE%) determination. The encapsulation efficiency (EE%) of MO extract in pharmacosomes was determined using an indirect ultraviolet spectrophotometric method. Briefly, nanoparticle suspensions (MO-PhS) were centrifuged at 15 000 rpm for 1.5 hours at 4 °C to separate unencapsulated phytochemicals. The absorbance of the collected supernatant was measured at 330 nm using a FLUOstar Omega microplate reader (BMG Labtech, Germany). This wavelength was selected to target a range of cytotoxic constituents, including phenolic acids and flavonoids, identified in the extract via GC-MS analysis, such as ferulic acid and chlorogenic acid. A calibration curve was constructed (y = 0.0017x + 0.1421, R² = 0.9905) to quantify the concentration of free phenolic acids and flavonoids, enabling indirect calculation of encapsulation efficiency. Encapsulation efficiency was calculated using the following equation:
where Total MO represents the initial concentration of Moringa oleifera extract, and Free MO corresponds to the unencapsulated fraction, as determined from the supernatant absorbance measurements.

3.4. Biological assessment assays

3.4.1. Antioxidant activity assessment. The antioxidant potential of the MO extract was evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Serial dilutions of the extract were prepared at concentrations ranging from 15.625 µg mL⁻¹ to 1 mg mL⁻¹ and mixed with an equal volume (1 : 1, v/v) of a 0.12 mM methanolic DPPH solution. The reaction mixtures were then incubated in darkness at room temperature for 30 minutes. Following incubation, the absorbance was measured spectrophotometrically at 517 nm (Cary 3500 UV-Vis Engine, Agilent Technologies, Pty Ltd, Mulgrave, Australia). The DPPH radical scavenging activity was calculated using the equation below, where A_β represents the absorbance of the DPPH methanolic solution (blank) and A_s denotes the absorbance of the sample mixture:

3.4.2. Biocompatibility assay against hFB cells. Normal human fibroblast (hFB) cell line was purchased from Rio de Janeiro Cell Bank (BCRJ, Brazil). Cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM), which comprised 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% antibiotic-antimycotic cocktail. All were purchased from PAN-Biotech (Germany). Cells were maintained at sub-confluency in humidified air containing 5% CO₂ at 37 °C. For the subculturing step, monolayer cells were treated with trypsin/EDTA and harvested at 37 °C. Cells were used upon reaching 75% confluency.⁵⁰

3.4.3. Cytotoxicity assay. The cytotoxicity of the tested samples was evaluated using the MTT assay (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide). This colorimetric method relies on the ability of mitochondrial dehydrogenase enzymes in viable cells to reduce the yellow MTT dye into insoluble dark blue formazan crystals. The amount of formazan formed after solubilization correlates directly with the number of metabolically active (viable) cells. Briefly, cells (1 × 10⁴ cells per well) were seeded into flat-bottom 96-well microplates and incubated overnight to allow attachment. Subsequently, cells were treated with 50 µL of varying concentrations of the tested samples, including MO extract, plain PhS, and MO-PhS nanoparticles, at final concentrations of 15.625, 31.25, 62.5, 125, 250, 500, and 1000 µg mL⁻¹. Treatments were performed in triplicate and incubated for 48 h at 37 °C in a humidified 5% CO₂ atmosphere. After incubation, the media were removed, and 50 µL of MTT solution (5 mg mL⁻¹) was added to each well and incubated for an additional 4 h under the same conditions. The resulting formazan crystals were solubilized by adding 150 µL of dimethyl sulfoxide (DMSO) per well. Plates were shaken gently at room temperature to ensure complete dissolution, followed by measurement of absorbance at 560 nm using a microplate ELISA reader (FLUOstar OPTIMA, BMG LABTECH GmbH, Ortenberg, Germany). All experiments were performed in triplicate, and average values were calculated.⁵¹ The percentage of relative cell viability was determined using the following equation:

The half-maximal inhibitory concentration (IC₅₀) was then calculated from the equation of the dose–response curve.

3.4.4. Flow cytometry analysis of cell cycle distribution. SH-SY5Y human neuroblastoma cells were seeded in 6-well plates and allowed to adhere for 24 h. Cells were then treated with MO extract and MO-PhS nanoparticles at their respective IC₅₀ concentrations, as previously determined by the MTT assay. Untreated cells served as the negative control. After 48 h of treatment, cells were harvested, washed with DPBS (Dulbecco's phosphate-buffered saline), trypsinized (0.05% trypsin–EDTA), and fixed in ethanol. Cell cycle analysis was performed using a Beckman Coulter Cell Cycle Analysis Kit (Cat. No. PN 4238055-CB), followed by propidium iodide staining. DNA content was analyzed using a CytoFLEX flow cytometer (Beckman Coulter Life Sciences, USA), and cell populations were quantified in G0/G1, S, and G2/M phases using the associated analysis software.⁵⁰

3.5. Statistical studies

The results were reported as mean ± standard deviation (SD). All formulations were prepared in triplicate. ImageJ software was used to analyze the particle size distribution of the obtained nanoparticles. Dose–response curves were generated using non-linear regression analysis with an asymmetric five-parameter logistic model: X = log(concentration), employing GraphPad Prism version 10.3.0 (507) for Windows (GraphPad Software, San Diego, CA, USA).

4. Results and discussion

4.1. GC-MS results and anticancer relevance of identified compounds against various tumors

The GC-MS analysis of the MO extract revealed a diverse array of phytochemicals (Fig. 1, S1 and Table 2), comprising several compounds with previously reported anticancer properties relevant to different malignancies. These results offer a compelling biochemical rationale for utilizing the extract in nanosystems aimed at treating neuroblastoma.

Fig. 1 Key identified compounds in the MO extract.

Table 2 List of bioactive compounds identified in the MO extract via GC-MS, with corresponding retention times (RT), peak numbers, and relative abundance^abc

No.	Compound name	MW (g mol^−1)	CAS number	RT (min)	Peak	Relative abundance (%)	Biological activity
a Retention times represent TMS-derivatized forms as identified by GC-MS analysis.b Some peaks correspond to multiple co-eluted compounds or broad compound classes (e.g., flavonoid, caffeic acid, and ferulic acid derivatives), as detected by spectral matching in the GC-MS analysis. Peak numbers reflect those assigned in the original chromatogram.c Molecular weights (MWs) and CAS numbers refer to the native, non-derivatized forms of the compounds. All MWs and CAS numbers were verified against authoritative chemical databases (PubChem, ChemSpider, Sigma-Aldrich). Approximate MWs (∼) are given where specific compound structures could not be assigned.
1	Glycerol	92.09	56-81-5	9.632	1	2.25%	Precursor for lipid synthesis; may influence tumor cell membrane dynamics
2	Malic acid	134.09	6915-15-7	11.189	3	4.13%	Showed anticarcinogenic effect on glioblastoma cells via the necrotic pathway and apoptotic properties on fibroblast cells
3	Quinic acid	192.17	77-95-2	13.249	13	10.6%	Has antioxidant activity that has shown anticancer properties through apoptosis-mediated cytotoxicity in breast cancer cells. Also, derivatives of quinic acid exhibited cytotoxic effects against glioblastoma cells by inducing apoptosis
4	Chlorogenic acid	354.31	327-97-9	13.249	13	10.6%	Induced differentiation and apoptosis in glioma cells; inhibited tumor growth in vivo
5	Myo-inositol	180.16	87-89-8	15.434	15	4.81%	Exhibited broad-spectrum anticancer activity; modulated PI3K/Akt pathway and inhibited tumor invasiveness
6	Uridine	244.20	58-96-8	16.401	32	22.16%	Inhibited hepatocellular carcinoma cell development by inducing ferroptosis
7	Shikimic acid	174.15	138-59-0	14.805	22	4.82%	Exhibited anticancer effects via suppressing migration and invasion, inducing apoptosis, and inhibiting proliferation in colorectal cancer cells. Additionally, derivatives of shikimic acid exhibited cytotoxic effects against liver cancer cells
8	Levoglucosan	162.14	498-07-7	12.533	8	0.84%	Derivatives showed cytotoxic activities against several sorts of cancers (lung, colon, and hepatocarcinoma cells)
9	Cinnamic acid	148.16	140-10-3	10.084	2	0.58%	Antioxidant phenolic acid. Induced cytostasis and differentiation in glioblastoma cells and other cancerous cells (melanoma, prostate and lung); inhibited tumor invasiveness
10	Feruloylquinic acid	368.3	62 929-69-5	13.249	13	10.6%	Limited direct evidence on anticancer activity; feruloylquinic acid and its derivatives exhibited antioxidant properties; showed neuroprotective effects
11	L-threonic acid	136.11	3909-12-4	11.48	4	0.29%	Limited direct evidence on anticancer activity; known for its antioxidant properties (vitamin C synergist)
12	Tartronic acid	120.06	80-69-3	10.084	2	0.58%	Limited direct evidence on anticancer activity; may inhibit lipid peroxidation
13	Glyceric acid	106.08	473-81-4	10.084	2	0.58%	Limited direct evidence on anticancer activity; involved in metabolic pathways. Glutathione precursor
14	L-(+)-Tartaric acid	150.09	87-69-4	10.084	2	0.58%	Limited direct evidence on anticancer activity; functions as a metal chelator
15	Serine	105.09	56-45-1	11.795	6	0.47%	Essential amino acid. Glutathione precursor
16	L-Methionine	149.21	63-68-3	12.814	10	12.23%	Sulfur-containing amino acid with antioxidant roles
17	Threonine	119.12	72-19-5	12.814	10	12.23%	Essential amino acid contributes to membrane integrity
18	Aromandendrene	204.35	489-39-4	9.632	1	2.25%	Anti-inflammatory terpene, with various anticancer properties
19	Nootkatone	218.34	4674-50-4	9.632	1	2.25%	A sesquiterpene compound. Exhibited antiproliferative activity in colorectal cancer cells. Showed promising neuroprotective effects while improving cognitive function in Alzheimer's disease models
20	Coumaroylquinic acid	338.31	32 451-86-8	13.249	13	10.6%	Polyphenolic ester; mitochondrial protective effects
21	Flavonoid derivatives	∼500–600	N/A	12.5–15	7, 18	29.02%	General class includes compounds with broad-spectrum anticancer activity; induce apoptosis in brain tumor cells
22	Caffeic acid derivatives	∼350–400	N/A	10–14	5, 9	2.78%	Antioxidant polyphenols with hydroxyl groups. Induced apoptosis in cancer cells; inhibit tumor growth
23	Ferulic acid derivatives	∼400–450	N/A	12–14	10, 13	22.83%	Neuroprotective and anti-inflammatory polyphenols. Inhibited angiogenesis and tumor growth; induced apoptosis in cancer cells
24	Fatty acid derivatives (oleic acid)	354.6	21 556-26-3	15.24	23	5.02%	Contribute to cell membrane stability and function. Also, oleic acid showed anticancer activity against different carcinomas

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Among the predominant compounds, uridine (22.16%) has demonstrated anti-tumor activity through its role in nucleotide metabolism, modulation of DNA/RNA repair mechanisms, enhancement of chemotherapeutic efficacy, and induction of ferroptosis. It is also involved in regulating tumor cell proliferation and survival, particularly in glioma and neuroblastoma models.^52–56

The extract also contained significant levels of phenolic acids, including feruloylquinic acid, chlorogenic acid, and quinic acid (10.6%). These compounds are recognized for their antioxidant properties and pro-apoptotic effects in cancer cells. Markedly, chlorogenic acid has been shown to induce apoptosis and cell-cycle arrest at the G2/M phase in glioma U373 cells, as well as suppress migration and invasion by modulating the SRC/MAPKs signaling pathway.⁵⁷

Myo-inositol (4.81%) and shikimic acid (4.82%) were also notable, both recognized for their cytotoxic effects and potential anticancer roles.^58–61 For instance, myo-inositol has demonstrated broad-spectrum anticancer potential by modulating critical pathways such as PI3K/Akt, disrupting growth factor signaling, and interfering with cytoskeletal dynamics. These effects collectively inhibit tumor progression and metastasis.⁵⁸

The extract also contained substantial amounts of flavonoid derivatives (29.02%) and ferulic acid derivatives (22.83%), which are well-documented for their cytotoxic effects, DNA damage induction, and selective apoptosis of tumor cells, including neuroblastoma and glioblastoma. These compounds are known to downregulate pro-survival genes and enhance ROS-mediated cytotoxicity in cancer cells.^62–70

Additionally, nootkatone (2.25%) was also detected. It is a promising sesquiterpene that exhibited antiproliferative activity in colorectal cancer cells.^71,72 Other minor but relevant compounds included caffeic acid derivatives, coumaroylquinic acid, and fatty acid derivatives such as oleic acid. Oleic acid has been shown to inhibit fatty acid and cholesterol synthesis in glioma cells, potentially contributing to the suppression of tumor growth.⁷³

Overall, the chemical profile revealed by GC-MS supports the anticancer potential of the MO extract and its integration into nanocarrier systems targeting several tumors, including neuroblastoma.

4.2. Morphological and physicochemical characterization of the nanoparticles

Ultra-high-resolution transmission electron microscopy (UHR-TEM) was employed to assess the morphological characteristics and confirm the nanoscale size of the developed formulations, alongside verifying the successful encapsulation of the MO extract. As illustrated in Fig. 2, TEM images for both plain pharmacosomes (PhS2) and MO-loaded pharmacosomes (PhS3) revealed well-dispersed, spherical nanostructures with smooth contours. The particle sizes observed were noticeably smaller, ranging from 41.07–190.12 nm for PhS2 and 26.05–211.08 nm for PhS3, and more uniform than those recorded by dynamic light scattering (DLS), suggesting the rare presence of aggregations and supporting successful formulation.

Fig. 2 Ultra-high-resolution transmission electron microscopy (UHR-TEM) images of (A) plain pharmacosome nanoparticles (PhS2) and (B) MO-loaded pharmacosome nanoparticles (PhS3). All images are shown with a 200 nm scale bar. Panel A displays unloaded nanoparticles with well-defined spherical to slightly oval morphology and minimal aggregation. Panel B illustrates MO-loaded pharmacosomes exhibiting enhanced structural compactness and denser appearance, indicative of successful MO extract encapsulation and integration within the lipid/chitosan matrix.

More importantly, MO-loaded pharmacosomes (PhS3) displayed a compact morphology with a more condensed structure, potentially indicating encapsulation and interaction between the MO extract and the lipid/chitosan matrix of the pharmacosomes. These visual cues also imply enhanced structural integrity, which is critical for biological applications, particularly in terms of cellular uptake and systemic circulation stability.^74,75

In contrast, DLS analysis yielded comparatively larger hydrodynamic diameters for all formulations, a discrepancy commonly attributed to the solvation layer and Brownian motion during measurement.^76,77 PhS1, with the highest lecithin content (4.5%), showed the largest Z-average values (255.67 ± 3.16 nm), the lowest surface charge (−29.93 ± 0.47 mV), and a comparable polydisperse profile (PDI 0.47 ± 0.019), suggesting that lecithin enrichment contributes to vesicle enlargement and reduced electrostatic repulsion. PhS2 and PhS3, with equal lecithin content but differing in MO presence in PhS3, demonstrated distinct size and charge profiles. PhS2 showed a more compact profile (average size of 190.4 ± 0.78 nm, PDI of 0.29 ± 0.04, and zeta potential of −58.73 ± 0.99 mV), while PhS3 presented a broader size distribution (280.93 ± 3.11 nm Z-average, 0.68 ± 0.08 PDI, and −55.73 ± 0.06 mV zeta potential). The high surface charge of PhS2 and PhS3 reflects strong electrostatic stabilization, likely imparted by both the phosphate groups of lecithin and the protonated amines of low molecular weight chitosan.^78,79

Interestingly, despite PhS3 (MO-loaded pharmacosomes) showing a broader size distribution by DLS, its corresponding TEM images revealed a more uniform and densely packed particle population. This disparity highlights the influence of formulation conditions (e.g., sonication, dilution, and dispersing medium) on hydrodynamic measurements, while emphasizing that TEM provides a more accurate representation of dry-state morphology.⁸⁰

From a functional standpoint, these nanoparticulate properties directly influence therapeutic performance. The reduced particle size and stable surface charge observed in MO-loaded pharmacosomes suggest efficient cellular internalization via endocytosis, particularly in neuroblastoma cells (SH-SY5Y), which possess high membrane fluidity and active endocytic pathways. Additionally, the negative surface potential aids in prolonged circulation by reducing opsonization and reticuloendothelial clearance.⁸¹ Notably, the observed nanometric size range (<300 nm), as confirmed by TEM, combined with the high zeta potential, indicates a strong potential for targeting tumor cells, including neuroblastoma. Encapsulation within the lipid matrix may further support receptor-mediated uptake and membrane fusion processes in neural cells, thereby enhancing selective cytotoxic delivery to tumor tissue.^82,83

4.3. Encapsulation efficiency of MO-loaded pharmacosomes (MO-PhS)

Encapsulation efficiency (EE%) of MO-PhS was quantified using an indirect UV-spectrophotometric method, as described in the methodology. The optimized formulation demonstrated a notably high EE% of 94.52 ± 2.79%, confirming the efficient entrapment of the constituents of MO extract within the pharmacosomal system.

This high EE% suggests a strong affinity between the Moringa phytochemicals and the lipid–chitosan matrix, likely facilitated by both hydrophilic and lipophilic interactions. The amphiphilic nature of the carrier system further contributed to this enhanced encapsulation, stabilizing a wide spectrum of phenolic compounds and flavonoids previously identified in the extract. This observation is supported by previous findings; Huang et al. (2017) demonstrated that liposomal encapsulation significantly improves the stability and retention of flavonoids such as quercetin and luteolin. Their study highlighted that interactions between flavonoids and the lipid bilayer enhance encapsulation efficiency and protect the compounds from degradation.⁸⁴

From a therapeutic perspective, this high encapsulation performance is crucial for maximizing bioavailability and ensuring efficient delivery to neuroblastoma cells. SH-SY5Y cells, characterized by their active endocytic behavior and high membrane fluidity, are likely to internalize these pharmacosomal nanoparticles effectively, enabling enhanced intracellular accumulation of cytotoxic phytoconstituents. Encapsulation also plays a protective role, shielding sensitive compounds from premature degradation and metabolic inactivation, an essential aspect in tumor targeting, where stability and sustained intracellular presence are required for cytotoxic efficacy. Sengupta et al. (2021) reviewed the benefits of liposomal encapsulation of phenolic compounds, emphasizing improved bioavailability, enhanced neuronal uptake, and protection from premature degradation.⁸⁵

4.4. FTIR spectroscopic characterization

The FTIR spectrum of the MO extract reveals several characteristic absorption bands indicative of its rich phytochemical composition. A broad O–H stretching vibration appears around 3300–3400 cm⁻¹, signifying the presence of hydroxyl groups from phenolic compounds and alcohols. Additionally, C–H stretching vibrations are observed near 2900 cm⁻¹, corresponding to aliphatic chains. The spectrum also displays a prominent C=O stretching band around 1700–1650 cm⁻¹, which can be attributed to carbonyl groups in flavonoids, aldehydes, or carboxylic acids. Aromatic C=C stretching vibrations emerge in the 1600–1500 cm⁻¹ region, while C–O–C and C–OH stretching bands appear between 1100 and 1000 cm⁻¹, indicating the presence of ethers and alcohols. These findings align with previous studies that have identified similar functional groups in Moringa leaf extracts.^86,87

The FTIR spectrum of the plain pharmacosomes (PhS) exhibits distinct bands characteristic of phospholipid structures. A broad band near 3300 cm⁻¹ suggests N–H or O–H stretching, possibly from amine or hydroxyl groups in the phospholipids. Strong C–H stretching vibrations are evident around 2920–2850 cm⁻¹, corresponding to the lipid alkyl chains. A significant peak at approximately 1735 cm⁻¹ indicates ester C=O stretching, a hallmark of phospholipid ester bonds. Notably, phosphoryl stretching (P=O) vibrations are observed near 1240 cm⁻¹, and phosphoester bond stretching (P–O–C) appears around 1092 cm⁻¹, confirming the presence of phosphate groups integral to the phospholipid structure.⁸⁸

The FTIR spectrum of the MO-loaded pharmacosomes (MO-PhS) combines features from both the Moringa extract and plain pharmacosomes spectra, indicating encapsulation of the extract within the nanoparticles. The broad O–H stretching band around 3300 cm⁻¹ is retained but exhibits slight shifts and broadening, suggesting hydrogen bonding interactions between Moringa phenolics and phospholipid molecules. The ester C=O stretching band near 1730 cm⁻¹ persists, albeit with some broadening, indicating overlapping carbonyl groups from both components. Additionally, the P=O and P–O–C stretching bands near 1240 cm⁻¹ and 1092 cm⁻¹, respectively, are present but show modifications in intensity and position, further supporting interactions between the extract and phospholipid matrix.⁸⁸ These spectral changes indicate that the phospholipid structure remains intact upon encapsulation, with possible hydrogen bonding or electrostatic interactions facilitating the incorporation of the Moringa extract (Fig. 3).

Fig. 3 FTIR spectra of Moringa extract (MO), plain pharmacosomes (PhS), and MO-loaded pharmacosomes (MO-PhS), highlighting key spectral bands. The shaded green region (3200–3500 cm⁻¹) corresponds to O–H stretching, showing broadening in MO-PhS indicative of hydrogen bonding between hydroxyl-rich phytoconstituents and lipid components. The red region (∼1700 cm⁻¹) marks the C=O stretching band, with notable shifts and intensity changes post-encapsulation due to overlapping signals from phenolic and ester carbonyls. The yellow (∼1240 cm⁻¹) and blue (∼1090 cm⁻¹) regions represent P=O and P–O–C stretching vibrations, respectively, which are retained in both PhS and MO-PhS, supporting the preservation of the phospholipid bilayer's integrity and suggesting potential electrostatic or hydrogen-bond interactions with the extract. These findings collectively confirm the successful incorporation of MO constituents into the pharmacosomal matrix.

4.5. Stability studies

Thermogravimetric analysis was conducted to evaluate the thermal stability of the MO, PhS, and MO-PhS (Fig. 4). The TGA curves exhibited distinct degradation patterns, reflecting the characteristic thermal behavior of each component. The MO curve showed a multi-step degradation process, initiating with a minor weight loss (11.3%) below 150 °C, attributed to the evaporation of residual water or volatile phytoconstituents, followed by a major decomposition event with 45.56% weight loss, occurring between 200 °C and 400 °C. This significant weight loss corresponds to the thermal breakdown of polyphenolic and other thermolabile constituents typically abundant in plant extracts.^89,90

Fig. 4 Thermogravimetric analysis (TGA) curves of Moringa extract (MO), plain pharmacosomes (PhS), and Moringa-loaded pharmacosomes (MO-PhS). The MO-loaded pharmacosomes exhibited enhanced thermal stability compared to free MO, as evidenced by the delayed onset of major weight loss and smoother degradation profile, but less stability as compared to unloaded pharmacosomes, attributing to the wide range of phytochemicals present in the MO extract, which differ in their polarity nature affecting the structure of the plain pharmacosomes.

The phospholipid profile presents in the plain pharmacosomes (PhS) demonstrated relatively better thermal stability, with the primary degradation beginning around 220 °C, with 7.04% weight loss, and extending up to 450 °C (31.32%). This major thermal event is associated with the decomposition of the phosphatidylcholine backbone and fatty acid chains, a finding consistent with previous reports on lecithin-based systems.^91,92 Notably, the MO-loaded pharmacosomes (MO-PhS) exhibited a delayed onset of thermal degradation and a smoother degradation curve compared to its individual components. The primary degradation step for MO-PhS shifted toward higher temperatures (onset 240 °C with 12.8% weight loss) while extended up to 475 °C with 32.8% weight loss, suggesting enhanced thermal stability resulting from successful interaction between MO and the pharmacosomal matrix.

This improvement in thermal resistance may be attributed to the formation of non-covalent interactions such as hydrogen bonding or electrostatic forces between the bioactive constituents of MO and the polar head groups of the phospholipid, particularly the phosphate moieties. Such interactions likely reduce the volatility and mobility of the extract within the lipid matrix, thereby providing a protective effect.⁹¹ Additionally, the more gradual weight loss curve observed in MO-PhS compared to MO suggests a controlled degradation behavior, which may reflect the encapsulation efficiency and the establishment of a stable nanocomplex. Similar stabilization effects have been reported in niosomes and other lipid-based nanocarriers encapsulating polyphenolic compounds.^91,93

Notably, the nanoformulations were stored at 4 °C for approximately one year, after which FTIR analysis and DLS measurements (size, PDI, and zeta-potential) were repeated, and the physical appearance was re-examined. No changes in the FTIR spectra or visible signs of sedimentation or aggregation were observed, indicating good chemical integrity and storage stability of the prepared nanoformulations.

4.6. In Vitro biocompatibility assessment on normal human fibroblasts

The biocompatibility of the developed pharmacosomal system was evaluated using normal human fibroblast (hFB) cells to assess the safety profile of the carrier and to determine whether nanoencapsulation confers selectivity toward cancer cells. Cell viability was assessed after exposure to plain PhS, free MO extract, and MO-PhS over the tested concentration range (Fig. 5).

Fig. 5 In vitro biocompatibility evaluation of plain PhS, MO extract, and MO-PhS in SH-SY5Y neuroblastoma cells. Cells were exposed to increasing concentrations for 48 h, and cell viability was assessed using the MTT assay. The results demonstrate no significant reduction in cell viability across the tested concentration range, confirming the cytocompatibility and biological safety of the samples. These findings indicate that the observed cytotoxic effects in MO-PhS are attributable exclusively to the encapsulated MO extract rather than the nanocarrier itself. Data represent mean ± SD of three independent experiments (n = 3).

Plain PhS exhibited negligible cytotoxicity toward hFB cells, with cell viability remaining above the accepted biocompatibility threshold across all tested concentrations. This confirms the intrinsic safety of the lecithin–chitosan pharmacosomal matrix and demonstrates that the carrier system itself does not induce nonspecific cytotoxic effects. The excellent biocompatibility observed is consistent with previous reports describing phospholipid-based nanocarriers and chitosan-containing systems as well tolerated by normal cells due to their biomimetic composition and biodegradability.^94,95 In contrast, exposure to the free MO extract resulted in a moderate, concentration-dependent reduction in fibroblast viability, reflecting the presence of bioactive phytochemicals with known biological reactivity. Polyphenolic acids, flavonoids, and fatty acid derivatives identified by GC-MS have previously been reported to exert mild cytostatic or oxidative effects in normal cells at elevated concentrations, particularly when administered in their free form without controlled release. However, this reduction in viability remained substantially lower than that observed in neuroblastoma cells, indicating partial selectivity toward malignant cells. Notably, PhS demonstrated improved biocompatibility compared to the free MO extract, with fibroblast viability consistently higher at equivalent concentrations. This finding suggests that nanoencapsulation effectively modulates the interaction between the phytochemicals and normal cells by controlling their release profile and limiting acute intracellular exposure. The pharmacosomal architecture likely reduces burst-related cytotoxicity while maintaining therapeutic activity in cancer cells, a feature commonly reported for lipid-based nanocarriers encapsulating natural anticancer agents.⁹⁴

The observed selectivity can be attributed to several factors, including the differential endocytic activity between normal fibroblasts and cancer cells, as well as differences in membrane composition and metabolic demand. Cancer cells such as SH-SY5Y neuroblastoma cells exhibit enhanced nanoparticle uptake and heightened sensitivity to redox imbalance, whereas normal fibroblasts possess more robust antioxidant defenses and lower proliferative stress, allowing them to better tolerate controlled phytochemical exposure.^94,95 Overall, these results confirm that the developed MO-PhS possess a favorable safety profile toward normal cells, while retaining strong cytotoxic activity against neuroblastoma cells. This selective behavior supports the suitability of the pharmacosomal system as a cancer-targeted nanocarrier and provides a critical safety foundation for subsequent mechanistic and therapeutic evaluations.

4.7. Cytotoxicity of MO-pharmacosomes in SH-SY5Y cells supported by antioxidant profiling

The cytotoxicity profiles of MO extract, plain PhS, and MO-PhS were assessed on SH-SY5Y neuroblastoma cells using the MTT assay after 48 h of exposure (Fig. 6). The IC₅₀ values, derived from the dose–response curves, revealed a significant enhancement in the biological activity of MO upon encapsulation.

Fig. 6 (A): Cell viability (%) of SH-SY5Y cells treated by Moringa oleifera extract (MO), plain pharmacosomes (PhS), and MO-loaded pharmacosomes (MO-PhS), assessed via the MTT assay after 48 h. (B) IC₅₀ values (µg mL⁻¹) of MO, PhS, and MO-PhS against SH-SY5Y cells. MO-PhS exhibited significantly enhanced cytotoxicity compared to free MO, with an IC₅₀ of 67.03 µg mL⁻¹, reflecting improved intracellular delivery and bioactivity. PhS alone showed negligible cytotoxicity (∼1000 µg mL⁻¹), confirming the biocompatibility of the nanocarrier. Data represent mean ± SD of three independent experiments (n = 3) (*p < 0.05; **p < 0.01 (0.0074)).

The MO extract exhibited moderate antiproliferative activity with an IC₅₀ of 695 µg mL⁻¹, aligning with prior studies where polyphenol-rich plant extracts demonstrated tumor-selective, dose-dependent effects on neuroblastoma and other cancer cell lines.^96–98 In contrast, MO-PhS achieved a markedly lower IC₅₀ of 67.03 µg mL⁻¹, indicating a roughly 10-fold increase in potency. Plain pharmacosomes, on the other hand, showed negligible cytotoxicity with an IC₅₀ exceeding 1000 µg mL⁻¹, thereby confirming the biosafety of the carrier system alone and attributing the observed cytotoxicity solely to the encapsulated phytoconstituents.

These findings correlate with the previously reported high EE% (94.52 ± 2.79%), which likely enhanced the bioavailability and intracellular delivery of the MO components. The amphiphilic nature of the phospholipid–chitosan matrix provided a dual-interaction platform capable of encapsulating both hydrophilic and lipophilic compounds, facilitating stable incorporation and controlled release of tumoricidal phytochemicals such as uridine, feruloylquinic acid, flavonoids, and caffeic acid derivatives.^{52,54,63,99,100}

In terms of antioxidant capacity, the MO extract demonstrated strong DPPH radical scavenging activity, with inhibition percentages of 65.23%, 68.16%, and 74.71% at concentrations of 15.625, 62.5, and 125 µg mL⁻¹, respectively. This supports the extract's redox-modulating potential and suggests its ability to combat oxidative stress often associated with tumor microenvironments.^101–104

Recent studies have highlighted the ability of the MO extracts to modulate redox-sensitive signaling pathways and apoptosis-related proteins in neuronal cells. For instance, Barinda et al. (2024)¹⁰² showed that MO leaf extract reduced ROS production and downregulated the expression of pro-apoptotic markers such as Bax and caspase-3 while upregulating phosphorylated Akt and CREB in SH-SY5Y neuroblastoma cells exposed to H₂O₂-induced oxidative stress. Although primarily evaluated for neuroprotective purposes, these molecular effects, particularly the modulation of apoptosis and redox homeostasis, also suggest potential relevance in targeting tumor cells.¹⁰² Supporting this, Al-Asmari et al. (2015)³⁶ demonstrated a significant cytotoxic effect of MO leaf and bark extracts against breast (MDA-MB-231) and colorectal (HCT-8) cancer cell lines, inducing apoptosis, cell cycle arrest at the G2/M phase, and inhibiting colony formation and motility.³⁶ These findings support the potential cytotoxic effect of the MO extract on SH-SY5Y neuroblastoma cells.

The nanoparticulate nature of the system further contributed to enhanced endocytic uptake by SH-SY5Y cells, which display active clathrin-mediated and caveolae-mediated endocytosis, making them particularly responsive to nanoscale formulations. These uptake mechanisms are particularly advantageous when using nanocarriers such as MO-PhS, as clathrin-mediated endocytosis ensures efficient cellular entry, while caveolae-mediated endocytosis facilitates cytoplasmic delivery with minimal lysosomal degradation, a critical feature for effective delivery of cytotoxic payloads.^105–107

The nanoscale size and surface properties of MO-PhS likely facilitated enhanced membrane penetration and endosomal escape, promoting higher intracellular delivery of phytochemicals. This is crucial for antitumor efficacy, enabling accumulation at the target site, induction of apoptosis, and disruption of proliferative signaling.^108,109

GC-MS profiling of the MO extract provided further evidence of its anticancer potential by revealing a rich pool of bioactive compounds. Uridine (22.16%) and myo-inositol (4.81%) have shown cytotoxic properties against different tumor tissues, modulating the P13K/Akt pathway (myo-inositol) and inducing ferroptosis (uridine), resulting in suppression of tumor invasiveness.^52,58,59 Polyphenolic acids such as feruloylquinic acid, chlorogenic acid, and caffeic acid derivatives possess potent antioxidant and pro-apoptotic activities in cancer models.^{57,64,99,110–119} Nootkatone, a sesquiterpene identified in the extract, has shown cytotoxic and anti-proliferative effects in colorectal cancer cell lines.^71,72 Flavonoid derivatives (29.02%) exhibit broad anticancer properties including ROS scavenging, cell cycle arrest, inhibition of angiogenesis, suppression of key tumor survival pathways, and even induction of apoptosis in tumor cells.^62,63,84,120 Other compounds such as ferulic and fatty acid derivatives further showed promising anticancer activities.^{65–70,121,122}

Encapsulation within pharmacosomes also acts as a protective barrier against premature degradation and first-pass metabolism, enhancing the pharmacokinetic and potentially targeting delivery profiles of the loaded phytochemicals. Similar delivery advantages have been reported with other plant-based nanoformulations targeting tumors.^123,124 Importantly, the observed cytotoxicity of MO-PhS at significantly lower doses, alongside the absence of toxicity from the carrier alone, strongly suggests that the effect is tumor-selective rather than nonspecific or cytolytic. Moreover, such nanoformulations have been shown to activate endogenous antioxidant response pathways, particularly nuclear factor erythroid 2-related factor 2 (Nrf2), which governs the expression of various cytoprotective, anti-apoptotic, and anti-tumorigenic genes in cancer systems.^125–128 This may further explain the enhanced bioactivity of MO-PhS compared to the free extract.

Taken together, MO-PhS demonstrated a promising antitumor profile against neuroblastoma cells, combining improved physicochemical properties, enhanced nanoscale uptake, high entrapment of cytotoxic phytochemicals, and a promising cytotoxic profile. The significantly reduced IC₅₀ reflects enhanced potency, delivery, and preservation of bioactivity, all of which justify further in vivo exploration of this delivery system for neuroblastoma therapy.

4.8. Flow cytometry analysis of cell cycle distribution

The effect of MO and MO-PhS on cell cycle progression in SH-SY5Y neuroblastoma cells is illustrated in Fig. 7. Cells were treated with MO and MO-PhS at their respective IC₅₀ concentrations determined by the MTT assay (695 µg mL⁻¹ and 67.03 µg mL⁻¹, respectively). Untreated control cells exhibited a typical proliferative profile, with the majority of cells residing in the G0/G1 phase (74.23%), followed by the S phase (24.30%) and a minimal fraction in the G2/M phase (1.47%). Treatment with MO extract resulted in a pronounced redistribution of the cell population, characterized by a reduction in the G0/G1 phase to 62.82%, accompanied by an increase in S phase cells to 31.89% and G2/M phase cells to 5.06%. This shift indicates that MO interferes with cell cycle progression, promoting accumulation of cells in phases associated with DNA synthesis and mitotic preparation. Notably, MO-PhS treatment induced more pronounced cell cycle modulation in terms of S-phase accumulation, with 61.64% of cells in G0/G1, 34.03% in the S phase, and 4.31% in the G2/M phase. Importantly, these effects were achieved at a nearly 10-fold lower concentration than that required for the free MO extract, highlighting the superior antiproliferative efficiency of the pharmacosomal formulation.

Fig. 7 Flow cytometric analysis of cell cycle distribution in SH-SY5Y neuroblastoma cells following treatment with free MO extract and MO-PhS as compared to control. (A) Cell cycle distribution analysis for control, MO, and MO-PhS; (B) cell cycle distribution for control; (C) cell cycle distribution for MO; (D) cell cycle distribution for MO-PhS.

Despite the similarity in the overall distribution pattern between MO and MO-PhS, the ability of MO-PhS to induce equivalent or stronger S- and G2/M-phase accumulation at a markedly reduced dose highlights the critical role of pharmacosomal encapsulation in enhancing intracellular delivery and bioavailability of MO phytoconstituents. This mirrors the cytotoxicity findings from the MTT assay, where MO-PhS exhibited an approximately 10-fold lower IC₅₀ compared to the free extract, confirming that the observed cell cycle effects are dose-efficient and formulation-driven rather than concentration-dependent.

The observed S-phase and G2/M accumulations are indicative of replication stress and checkpoint activation, which are well-established mechanisms leading to growth arrest and apoptosis in cancer cells. Such cell cycle perturbations are commonly associated with DNA damage, inhibition of DNA synthesis, and impaired mitotic progression.^129,130 The enhanced S-phase accumulation observed with MO-PhS suggests a more effective disruption of DNA replication dynamics, consistent with increased intracellular availability of active phytochemicals.

Lipid-based pharmacosomes closely mimic biological membranes, facilitating efficient cellular internalization and sustained intracellular exposure.¹³¹ This enhanced delivery likely amplifies the effects of key MO-derived compounds, such as flavonoids, phenolic acids, and isothiocyanate-related constituents, which are known to interfere with cyclin-dependent kinases, DNA replication machinery, and cell cycle regulatory proteins.^132,133 Importantly, the modulation of cell cycle distribution observed here does not reflect nonspecific cytotoxicity of the carrier system, as plain PhS were shown to be biocompatible in parallel assays. Instead, the data support a mechanism in which pharmacosomal encapsulation potentiates the anticancer activity of the MO extract by improving uptake, stability, and intracellular retention.

Overall, these findings demonstrate that both MO and MO-PhS exert antiproliferative effects in SH-SY5Y neuroblastoma cells through cell cycle arrest; however, MO-PhS achieves these effects at substantially lower concentrations, providing strong mechanistic support for the enhanced cytotoxic efficacy of the nanoformulated system. This dose-efficient cell cycle modulation further validates MO-PhS as a promising nanoplatform for neuroblastoma therapy.

5. Conclusion and future perspectives

This study demonstrates that pharmacosome-based nanocarriers represent an effective strategy to enhance the anticancer potential of Moringa oleifera extract against neuroblastoma. The successful incorporation of MO into phospholipid-chitosan pharmacosomes resulted in a stable nanoscale delivery system with high entrapment efficiency, favorable surface properties, and excellent biocompatibility. Importantly, pharmacosomal encapsulation significantly improved the cytotoxic activity of MO against SH-SY5Y neuroblastoma cells (10-fold increase), which was further supported by cell cycle analysis revealing pronounced arrest in S and G2/M phases, indicative of disrupted DNA replication and proliferative signaling. Crucially, these comparable or enhanced cell cycle effects were observed at substantially lower MO concentrations when delivered via pharmacosomes, reinforcing the formulation's superior intracellular delivery efficiency and therapeutic potency. Additionally, antioxidant evaluation by the DPPH assay further highlighted the extract's potent free-radical scavenging capacity, which supports its therapeutic potential.

The enhanced biological performance of MO-loaded pharmacosomes can be attributed to improved cellular internalization, sustained intracellular availability of phytochemicals, and strong drug–lipid interactions inherent to pharmacosomal systems. Unlike free extracts, which are often limited by poor solubility and instability, pharmacosomes provide a structurally integrated platform that preserves bioactivity while minimizing nonspecific toxicity, as evidenced by the low cytotoxicity of the plain carrier.

Despite these promising results, several challenges must be addressed to facilitate clinical translation. Scale-up of pharmacosome production requires optimization to ensure batch-to-batch reproducibility and cost-effectiveness, particularly when using complex plant extracts with variable composition. Regulatory approval may also present challenges, as botanical-based nanomedicines must satisfy stringent requirements regarding standardization, safety, and quality control. Furthermore, comprehensive in vivo evaluation, pharmacokinetic profiling, and long-term toxicity studies will be essential to validate therapeutic efficacy and safety in clinically relevant models.

Overall, this work provides a strong proof-of-concept for the use of pharmacosome-based nanocarriers to repurpose medicinal plant extracts for cancer therapy. With further optimization and preclinical validation, MO-loaded pharmacosomes may offer a viable and innovative approach for the treatment of neuroblastoma and other brain-related malignancies.

Conflicts of interest

The authors declare no competing interests.

Data availability

The data supporting this article have been included inside the manuscript.

Supplementary information (SI): the GC-MS chromatogram conducted on MO extract. See DOI: https://doi.org/10.1039/d5na00473j.

Acknowledgements

The current work was funded by a grant from the American University in Cairo to Distinguished University Prof. Hassan Azzazy.

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