Abstract
Lycopene is the highest concentration of red, hydrophobic carotenoids in various fruits and vegetables. Because of low solubility and poor bioavailability, a delivery system must be utilized to have a prolonged target therapeutic site. The current research is focused on assessing the potential antioxidant and anticancer activity of Lycopene Cubosomal Nanoparticles against the HT-29 colon cancer cell line. The nanoformulation was ensured by various characterization methods, including FTIR, TEM, SEM, particle size, and zeta potential. The structure of Lycopene was energy-minimized and was docked into the ATP-binding pocket using SwissDock with AutoDock Vina as backend. DPPH and ABTS were used for the presentation of antioxidant activities. In addition, anticancer activity of the compounds was tested by MTT viability test. Cell cycle and annexin-V/PI double staining were also investigated by flow cytometry. Further, gene expression and protein content of different markers PI3K, AKT, mTOR, Caspase-3, and Bcl-2 were analyzed by qRT-PCR and ELISA methods. The results presented that Lycopene Cubosomal Nanoparticles are more active than Lycopene as regards antioxidant and anticancer activity. Additionally, PI3K, AKT, mTOR, and Bcl-2 gene expression and content were decreased, and the gene expression and content of Caspase-3 were increased in both treatments, particularly pointing towards Lycopene Cubosomal Nanoparticles. Highly soluble Lycopene Cubosomal Nanoparticles were revealed to be more active than weakly soluble Lycopene alone, in that activity was enhanced over weakly soluble Lycopene alone, and Lycopene Cubosomal Nanoparticles were found to be more active than Lycopene alone as an antioxidant and anticancer agent.
Introduction
Cancer is one of the primary worldwide causes of mortality, with an estimated 19.3 million new cases and about 10 million deaths from cancer in the year 2020 alone1. The disease is marked by the uncontrolled growth of abnormal cells that invade adjacent tissues and metastasize to other organs2. Chemotherapy is associated with symptoms such as nausea, sores on the mouth, and low blood counts since both cancerous and normal dividing cells are impaired. It also results in long-term problems such as damage to the nerves and difficulty thinking, ultimately making survival more difficult3,4. Accordingly, there is an urgent need for anticancer strategies to overcome chemotherapy-induced adverse effects without affecting or enhancing the anticancer efficacy of these drugs. In the last few years, natural antioxidants have been under severe scrutiny for their potential to negate the deleterious effects of chemotherapeutic agents. Among them, lycopene, a potent carotenoid found in tomatoes, watermelons, and other red fruits, has been well investigated for its antioxidant, anti-inflammatory, and anticancer properties5,6,7,8. Lycopene possesses a conjugated double bond chain, whose antioxidant activity is very high. Such a system enables lycopene to protect cells from the damaging effects of ROS and resist oxidative degradation of cellular components5. Moreover, studies have demonstrated that lycopene can also activate GPx and SOD enzymes and improve the resistance of cells to oxidative stress conditions9. Such antioxidant properties are attributed to the protective effects of lycopene on lipids, proteins, and cellular DNA from oxidative degradation, which contributes to the development of cancer and cardiovascular diseases, a fact that is well linked to the prevention and treatment of various diseases caused by oxidative stress conditions10. Moreover, lycopene intake has also been associated with reduced levels of relative oxidative stress biomarkers in clinical studies among human subjects, as suggested by its ability to promote redox balance in cells. Lycopene’s poor bioavailability (1.85 ± 0.39%) in the experimental rat model and poor absorption through the GIT, as well as instability in its natural form, has also restricted its use as a therapeutic agent and led to the requirement for the design of new delivery systems that will improve its utilization and efficacy as a potential therapy for the prevention and treatment of serious diseases like cancer and cardiovascular diseases6,11–13.
Nanoformulation technology is a groundbreaking improvement in increasing the target delivery and bioavailability of bioactive molecules14. Encapsulation of lycopene into nanocarriers increases its delivery to the tissues, stability, and solubility, hence optimizing its therapeutic efficacy2. As our past work has shown, antioxidant nanoformulations can neutralize drug-induced toxicities through increased cell uptake and alteration of the drug mechanisms of oxidative stress6,15,16. An investigation by Singh et al. aimed to develop an ideal lycopene nanostructured lipid carrier (NLC) that enhances absorption after consumption17. Another study reported that comparing lycopene-loaded polyelectrolyte complex nanoparticles (PEC NPs) to free lycopene, the former may be an effective lycopene carrier that improved the encapsulated lycopene’s water dispersibility, storage stability, antioxidant capacity, and sustained release ability in aqueous environments18. These advances provide a great justification for exploring the potential of lycopene nanoformulations as an alternative to well-known antioxidant pharmaceuticals.
Cubosomes are small liquid-crystal nanoparticles (100–500 nm) composed of special lipids forming honeycomb-like three-dimensional structures with hydrophilic and hydrophobic regions19. Unlike liposomes, they do not possess membrane bilayers, and the composition includes the blending of lipid bilayer structures with water, which allows for prolonged drug release19,20. Cubosomes exhibit high drug-loading capacity, drug-retention ability, and stability, which are mainly attributed to the amphiphilic nature of their constituent lipids21,22,23,24. The main component is glyceryl monooleate due to its excellent biocompatibility and biodegradability. Among common stabilizers, poloxamer 407 is most frequently used for preventing its aggregation and enhancing the stability of dispersion25,26. In view of these aspects, lycopene cubosomal nanoparticles can remarkably increase the stability and bioavailability of lycopene, protecting it from degradation and improving aqueous solubility. The cubosomes possess a liquid-crystalline state with sustained drug release and improved tissue uptake, as well as a nanoscale size with cellular uptake facilitation and a biocompatible nature, possibly inhibiting adverse effects due to oxidative stress. Nonetheless, a major disadvantage of cubosomal formulations of lycopene includes complicated preparation, scalability, stability, and increased production cost19,26.
In light of the benefits derived from delivery systems via nanoformulations, lycopene has been found to exhibit potent antiproliferative and apoptotic effects on various models of cultured cancer cells, and nanoformulations of lycopene were found to be much more cytotoxic than free lycopene. Free lycopene at concentrations of 10–20 µM significantly lowered HT-29 cell viability (IC₅₀ = 7.89 µM at 24 h)27, and antiproliferative effects were seen at concentrations of 8 µM with increased levels of caspase-3/PARP expression in HT-29 cells by Ataseven et al. (2023)28. Lycopene at concentrations of 1–5 µM also induced apoptosis in HT-29, HT-84, and MCF-7 cells after 48 h of treatment29. In fact, nano-formulations were found to be much more effective for lycopene delivery, with nanolycopene (NLY2) causing decreased cell viability to only 35–40% compared to free lycopene at 60–70% and causing over 70% cell death in B16 melanoma cells30. In the study on polymeric & gold nanoparticles-based lycopene formulation on prostate & colorectal cancer cells, the result revealed the dual cytotoxic & apoptotic activity with strong synergy31,32. At the molecular level, the role of lycopene in modulating activities like proliferation, migration, and invasiveness of cancer cells, associated with cancer, and reduction in oxidative stress has been demonstrated to influence cancer positively. It inhibits the expression of MMP-2, MMP-7, MMP-9, Sp1, IGF-1R, and VEGF, but increases E-cadherin, connexin 43, nm23-H1, TIMP-1, and TIMP-2. Its anticancer properties are also related to modulating proteins, like caspase-3, caspase-8, Bax, and ratios of Bax/Bcl-2 and Bax/Bcl-xL, which are associated with apoptosis33,34,35. In colorectal cancer models, lycopene has also demonstrated chemopreventive potential through inhibition of the PI3K/Akt signaling pathway in HT-29 cells33,34,35.
This study aimed to provide a foundation for developing novel nanoformulation-based therapeutic strategies that enhance the bioavailability of lycopene, positioning it as a promising natural antioxidant and anticancer agent.
Results
Characterization of Lyc-Cub-NPs
The average hydrodynamic diameter of lycopene cubosome was 154.37 ± 1.16 nm, PDI was 0.21 ± 0.025, and zeta potential was − 30.58 ± 2.85mV. The drug content of Lyc-Cub-NPs was estimated spectrophotometrically via a suitable dilution of colloidal dispersion in n-hexane. The UV-visible scanning of lycopene (from 200 to 600 nm) utilizing n-hexane as a blank illustrated the characteristic triple peaks at λ max 444, 471, and 503 nm. The triple peaks of lycopene can be considered as a characterization tool which can be employed to differentiate between lycopene and other major carotenoids such as lutein, β-carotene, and α-carotene (Fig. 1A). The absorbance of Lyc-Cub-NPs was determined, and the average drug content was 3.3 ± 0.23 mg/ml, and the percent entrapment efficiency (EE%) was 83.3 ± 5.67% (Table 1). According to the results shown in Table 1; Fig. 1B, the Lyc-Cub-NPs showed enhanced solubility in comparison to Lycopen (sparingly soluble in water); more than 75% of lycopene was detected after 15 min. The calculated dissolution efficiency after 60 min was 67.77 ± 0.68%.
A The UV-Visible scanning of Lycopene and Lyc-Cub-NPs (employing n-hexane as a blank), X-axis represents wavelength (nm), and Y-axis represents absorbance. The triple peaks (λ max 444, 471, and 503 nm) of lycopene can be considered as a characterization tool that can be employed to differentiate between lycopene and other major carotenoids, such as lutein, β-carotene, and α-carotene. B The percent lycopene released from Lyc-Cub-NPs at different time intervals (15, 30, 45, and 60 min), X-axis represents time (min), and Y-axis represents the % lycopene released from Lyc-Cub-NPs. Results are expressed as a mean ± SD (n = 3).
SEM demonstrated the surface structure of Lyc-Cub-NPs (magnification power 50000), and the surface scanning showed minute spherical particles (Fig. 2A). The TEM illustrated the hexagonal structure of the cubosome (honeycomb structure) (Fig. 2B). FTIR analysis of lycopene revealed characteristic absorption bands corresponding to functional group vibrations. Peaks observed at 2925.21 cm⁻¹ and 2854.36 cm⁻¹ were attributed to C–H stretching vibrations (symmetric and asymmetric modes of aliphatic CH bonds). The absorption band at 1637.37 cm⁻¹ was assigned to the C = C stretching of the trans-configured polyene chain, while the peak at 1020.55 cm⁻¹ corresponded to C–H bending vibrations (trans-configuration). Additionally, bands below 1000 cm⁻¹; a characteristic forked peak in the form of Walrus teeth (671.87 cm⁻¹ and 466.59 cm⁻¹) were associated with deformation vibrations of the Lycopene skeleton. A broad absorption band at 3452.66 cm⁻¹ region indicated O–H stretching vibrations, likely due to adsorbed water molecules (Fig. 3A). Comparative analysis demonstrated a near-complete overlap of Lycopene-specific peaks with the cubosome matrix, suggesting successful encapsulation. Notably, the disappearance of peaks at 1020.55 cm⁻¹, 671.87 cm⁻¹, and 466.59 cm⁻¹ further supports the entrapment of Lycopene within the lipid bilayer structure of the cubosomes, leading to restricted vibrational modes (Fig. 3B).
SEM of Lyc-Cub-NPs (A) and TEM of Lyc-Cub-NPs (B).
FTIR of Lycopene (A) and Lyc-Cub-NPs (B).
Molecular docking
AutoDock Vina generated multiple high-quality binding poses, with calculated affinities for Lycopene ranging from − 5.443 to − 2.888 kcal/mol. The best-ranked pose placed the lycopene molecule deep within the hydrophobic ATP-binding cleft of PI3Kα, in close proximity to key residues (e.g., Met772, Trp780, Val850), consistent with established inhibitor binding regions. PLIP analysis highlighted extensive hydrophobic interactions, verifying the structural accommodation of Lycopene’s flexible, zig-zag backbone within the pocket (contact distances 3.58–3.85Å). The interaction profile sustains the hypothesis that lycopene can bind directly and stably to PI3Kα, potentially disrupting its enzymatic activity and downstream signaling in cancer cells (Fig. 4; Tables 2 and 3).
Molecular Docking of Lycopene with PI3K-α in HT-29 Colon Cancer Cells.
Antioxidant activity estimation
Antioxidant activity was determined based on the DPPH radical scavenging activity (Fig. 5A). Treatment with Lycopene and Lyc-Cub-NPs was determined to possess concentration-dependent DPPH scavenging activity. Lyc-Cub-NPs possessed higher activity than Lycopene alone. Ascorbic acid, which is a positive control, had an IC50 (IC50, with 50% scavenging) of 3.16 µg/ml, whereas Lycopene and Lyc-Cub-NPs had mean scavenging concentrations of 42.61 and 8.43 µg/ml, respectively (Fig. 5A). In the same manner, the results of the ABTS experiment were also found to agree with those of the DPPH assay, and that higher antioxidant effect was observed for Lyc-Cub-NPs compared to Lycopene alone (Fig. 5B). IC50 of Lycopene and Lyc-Cub-NPs in the ABTS scavenging experiment were found to be 58.28 and 27.47 µg/ml, respectively. IC50 of gallic acid, a positive control, was 3.56 µg/ml (Fig. 5B).
Determination of antioxidant activity using A DPPH and B ABTS scavenging methods for Lycopene and Lyc-Cub-NPs. Results are expressed as a mean ± SD (n = 3). *means significant versus the control group. Each group differed significantly from the others at p ≤ 0.05. Control was ascorbic acid with an IC50 of 3.16 µg/ml for DPPH assay and Gallic acid with an IC50 of 3.56 µg/ml for ABTS assay.
Viability test of lycopene and Lyc-Cub-NPs against the HT-29 colon cancer cell line
A viability assay of Lycopene and Lyc-Cub-NPs was established against the HT-29 Colon Cancer Cell line after 48 h incubation. The viability and inhibitory effect of Lycopene and Lyc-Cub-NPs are shown in Fig. 6A and B with IC50 values of 95.25 and 54.04 µg/ml, respectively. Lyc-Cub-NPs showed the lowest viability and highest inhibition Fig. 6A and B.
Effect of Lycopene and Lyc-Cub-NPs on A Viability and B Inhibition of HT-29 Colon cell line using MTT viability test (incubation for 48 h) with IC50 = 95.25 and 54.04 µg/ml, respectively. Data are expressed as mean ± SD, n = 3.
Cell-cycle analysis
Flow cytometric cell-cycle analysis of Lycopene and Lyc-Cub-NPs is depicted in Fig. 7, varying cell-cycle phases (G0/G1, S, and G2/M) on the exponential growth phase of the HT-29 Colon Cancer Cells (incubation for 48 h). Both treatments depicted a G0/1-phase cell-cycle arrest with special reference to Lyc-Cub-NPs. Lyc-Cub-NPs caused extensive G0/1-phase cell-cycle arrest from 34.6% in untreated cancer cells to 85.7% in treated cells.
Annexin V/PI double-staining
The apoptotic cells that were impacted included Lycopene and Lyc-Cub-NPs that were revealed by annexin-V/PI double staining during the exponential growth phase of HT-29 Colon Cancer Cells (48 h incubation), as shown in Fig. 8. Both treatments showed an increase in apoptotic cells with particular reference to Lyc-Cub-NPs. Lyc-Cub-NPs revealed a significant increase in apoptotic activity from 1.5% in untreated cancer cells to 27.3% in treated cells.
Estimation of HT-29 Colon Cancer Cells cell-cycle distribution in untreated cancer cells (control) (A), after treatment with Lycopene (B), and after treatment with Lyc-Cub-NPs (C), which was analyzed using flow cytometric analysis based on IC50 concentrations detected by MTT assay (incubation for 48 h).
Estimation of apoptotic cell populations of HT-29 Colon Cancer Cells in untreated cancer cells (control) (A), after treatment with Lycopene (B), and after treatment with Lyc-Cub-NPs (C), determined by annexin V/PI double staining based on IC50 concentrations detected by MTT assay using flow cytometry (incubation for 48 h). The lower-right quadrant showed early cells in apoptosis.
Effect of lycopene and Lyc-Cub-NPs on PI3K, AKT, mTOR, caspase-3, and Bcl-2 gene expression in HT-29 colon cancer cells
PI3K, AKT, mTOR, Caspase-3, and Bcl-2 gene expression was exposed by qRT-PCR in the HT-29 Colon Cancer Cells with the IC50 concentration achieved through MTT cell viability test for Lycopene and Lyc-Cub-NPs (IC50 = 95.25 and 54.04 µg/ml, respectively, incubated for 48 h) (Fig. 9). Compared to the untreated (Control) group, the HT-29 cell line treated with Lycopene and Lyc-Cub-NPs showed downregulation PI3K (33% and 57%, respectively), AKT (31% and 57.13%, respectively), mTOR (37.62% and 53.47%, respectively) and Bcl-2 (36.79% and 59.43%, respectively) gene expression. Contrariwise, Lycopene and Lyc-Cub-NPs treatment upregulated the expressions of Caspase-3 (31.68% and 55.45%, respectively) in comparison to Control. Moreover, treatment with Lyc-Cub-NPs showed more noticeable results (Fig. 9).
Effect of lycopene and Lyc-Cub-NPs on PI3K, AKT, mTOR, caspase-3, and Bcl-2 markers in HT-29 colon cancer cells
PI3K, AKT, mTOR, Caspase-3, and Bcl-2 protein content was revealed by ELISA kits in the HT-29 Colon Cancer Cells with the IC50 concentration achieved through MTT cell viability test for Lycopene and Lyc-Cub-NPs (IC50 = 95.25 and 54.04 µg/ml, respectively, incubated for 48 h) (Fig. 10). In comparison to the untreated (Control) group, the HT-29 cell line treated with Lycopene and Lyc-Cub-NPs showed Lower PI3K (69.67% and 82.9%, respectively), AKT (62.3% and 93.7%, respectively), mTOR (62.5% and 92.7%, respectively) and Bcl-2 (26% and 66.81%, respectively) content. Conversely, Lycopene and Lyc-Cub-NPs treatment amplified the expressions of Caspase-3 (2.08-fold and 3.59-fold, respectively) in comparison to Control. Moreover, treatment with Lyc-Cub-NPs showed more pronounced results (Fig. 10).
Effect of Lycopene and Lyc-Cub-NPs on the gene expression of A PI3K, B AKT, C mTOR, D Caspase-3, and E Bcl-2 in the HT-29 cell line using qRT-PCR kits on the IC50 concentration detected by MTT viability test (IC50 = 95.25 and 54.04 µgml, respectively, incubation for 48 h). Data were expressed as mean ± SD (n = 3). *means significant versus the control group, and #means significant versus the Lycopene group. Control: cancer cell without any treatments. Each group differed significantly from the others at p ≤ 0.05.
Effect of Lycopene and Lyc-Cub-NPs on the protein content of A PI3K, B AKT, C mTOR, D Caspase-3, and E Bcl-2 in the HT-29 cell line using ELISA kits on the IC50 concentration detected by MTT viability test (IC50 = 95.25 and 54.04 µgml, respectively, incubation for 48 h). Data were expressed as mean ± SD (n = 3). *means significant versus the control group, and #means significant versus the Lycopene group. Control: cancer cell without any treatments. Each group differed significantly from the others at p ≤ 0.05.
Discussion
Lycopene is the most abundant red, hydrophobic carotenoid in many fruits and vegetables36. The best dietary source of lycopene is tomatoes. So, regular consumption of tomatoes provides sufficient amounts of lycopene for all body tissues37. The metabolism of lycopene is carried out mainly in the liver, though many enzymatic reactions are required to convert lycopene into an active form. When plasma levels of lycopene are decreased, the stored lycopene in the liver is liberated to adjust the normal plasma levels again38. The high lipid content tissues, as adipocytes, kidneys, ovaries, and prostate, need significant amounts of lycopene39. Lycopene is reported to be essential for preventing cancer, heart disease, high blood pressure, and inflammatory and neurological illnesses37,40,41,42,43.
Cubosomes were selected as a drug delivery system to overcome the limited aqueous solubility (log P = 17.64) and stability of lycopene44,45. Their lyotropic liquid-crystalline structure prevents drug precipitation, facilitates solubilization in the gastrointestinal tract, provides mucosal adhesion, and enables controlled degradation and release, thereby enhancing intestinal absorption and systemic bioavailability46,47,48. In contrast to liposomes and SLNs, cubosomes possess better loading and release properties owing to their unique structures, which are comprised of a bicontinuous cubic phase with lipid bilayers and possess two non-intersecting channels of water with a high drug loading capability for lipophilic drugs50,51. The results proved the formation of cubosome nanoparticles that were monodisperse and stable, as confirmed by characterizing their size, polydispersity index, and zeta potential, which were 154.37 ± 1.16 nm, 0.21 ± 0.025, and − 30.58 ± 2.85 mV, respectively, as confirmed with TEM and SEM results52,53. The TEM examination is based on electron transmission through a very thin sample layer (very diluted colloidal sample), which gives a 2-dimensional image characterized by higher magnification and resolution than a scanning electron microscope (SEM). SEM analysis verified the monodisperse nature of the cubosome nanoparticles. The SEM represents a three-dimensional image of a relatively thick sample. The sample must be dried and coated with gold before examination, which could drastically affect the shape and size of the particles (shrinking of the vesicles)54. The FTIR results showed altered bonding patterns upon entrapment with lycopene55, and dissolution profile experiments performed under sink conditions showed increased solubility with 69.77 ± 0.68%. Based on the lipophilic nature of lycopene, the sink condition was confirmed by the utilization of 500 ml of 0.5% sodium lauryl sulphate. The dissolution test was performed according to the previously published method56,57. The dissolution efficiency is considered a measuring tool to estimate the overall dissolution behaviour of the cubosome in comparison to the maximum possible dissolution within a certain time; DE% value lies between 0 and 100%. A DE% value close to 100% indicates complete and faster dissolution. The results illustrated enhanced dissolution of lycopene with DE% 69.77 ± 0.68% after 60 min. The high DE% can be explained by utilization of poloxamer 407 and polyvinyl alcohol as non-ionic surface active agents, which enhance the apparent aqueous solubility of lycopene. Our results illustrated a high EE% (83.3 ± 5.67) which which can be explained by a higher concentration of GMO, which provides a lipid matrix capable of entrapping lycopene effectively (GMO: lycopene ratio is 10:1)58.
Molecular docking using AutoDock Vina showed that lycopene would associate in the hydrophobic ATP-binding pocket in PI3Kα (binding scores − 5.443 to −2.888 kcal/mol), in close association with critical residues (Met772, Trp780, and Val850). PLIP validation indicated strong hydrophobic interactions (3.58–3.85 Å), validating the hypothesis that lycopene can directly and stably bind to PI3Kα, potentially disrupting its enzymatic activity and downstream signaling in cancer cells, as supported by the interaction profile59. These findings emphasize that the cubosomal encapsulation of lycopene not only improves its physicochemical properties but could potentially increase its anticancer potency through improved molecular interactions.
The antioxidant activity of lycopene and lycopene cubosomal nanoparticles, as elicited by DPPH and ABTS radical scavenging assays, agrees with previously reported high radical scavenging activity of lycopene. That strong antioxidant potential is due to its structure, which is characterized as a highly conjugated polyunsaturated hydrocarbon with eleven conjugated double bonds and has high singlet oxygen quenching capacity60. Our results agree with earlier reports where lycopene has been described to raise potent anti-oxidative stress activity through activation of multiple molecular mechanisms, including enhancement of antioxidant enzymes CAT, GSH, and SOD, NF-κB signaling inhibition, reduction of MAPK phosphorylation, and modulation of Nrf2 activity. Further, lycopene decreases the amount of pro-inflammatory cytokines, which include TNF-α and IL-1β, and improves inflammatory status by modulating glial markers, such as GFAP and IBA-161,62,63,64,65,66.
The anticancer action of lycopene and lycopene cubosomal nanoparticles (Lyc-Cub-NPs) was analyzed using viability assays, analysis of cell cycles, and Annexin/PI staining. The viability test also confirmed that it possessed significant anticancer action against the HT-29 Colon cancer cells, showing a significant decrease in viable cells, which was found to be most pronounced in Lyc-Cub-NPs. The mechanism of the anticancer effect of lycopene is related to multiple pathways: the antioxidant and anti-inflammatory properties of the compound and the inhibition of the proliferation of tumor cells. Lycopene showed significant antioxidant activity, surpassing that of β-carotene and α-tocopherol67, and decreased oxidative stress due to the scavenging action of ROS63. Earlier studies also demonstrated that lycopene regulated mechanisms related to cell injury and death, wherein it induced cell repair and apoptosis due to chronic oxidative stress68. Moreover, marked inhibition of HT-29 cells following lycopene treatment was evident due to the enhancement of pro-apoptotic proteins such as cleaved caspase-3, BAX, cleaved PARP, the transfer of cytochrome C into mitochondria, as well as levels of 8-oxo-dG, indicating that it can be used as a new drug molecule for colon cancer treatment28.
Cell cycle analysis of lycopene and Lyc-Cub-NPs by flow cytometry revealed that all three treatments significantly arrested the division of HT-29 cells at 48 h, establishing their efficacy in inhibiting cell growth. Interestingly, Lyc-Cub-NPs showed significant G0/G1 phase arrest, resulting in increased G0/G1 cells from 34.6% in control cells to 85.7% in treated cells, establishing the superiority of the cubosomal formulation. This was also validated by Annexin-V/PI staining, which showed increased levels of apoptosis by all three treatments. Notably, Lyc-Cub-NPs showed significantly increased levels of apoptosis, increasing from 1.5% in control cells to 27.3% in treated cells. All these results are consistent with earlier studies establishing that cell cycle arrest at key checkpoints and induction of apoptosis are efficient methods for cancer treatment69,70,71,72,73. However, the significantly higher G0/G1 cell cycle arrest and levels of apoptosis in the current study indicate that cubosomal formulation significantly improves the anti-cancer properties of lycopene over its free counterparts. In conclusion, the current study not only extends earlier studies investigating plant-derived anti-cancer agents but also verifies that lycopene-loaded cubosomes not only inhibit HT-29 cell proliferation but also significantly enhance cell cycle arrest and apoptosis, establishing their potential as an efficient anti-cancer agent against colon cancer.
To confirm the anti-cancer activity of lycopene and Lyc-Cub-NPs, mRNA and protein levels of autophagy markers, PI3K, AKT, and mTOR; apoptosis marker Caspase-3; and antiapoptotic marker Bcl-2 were measured using qRT-PCR and ELISA. All treatments showed a downregulation of PI3K, AKT, mTOR, and Bcl-2 protein expression, as well as an increase in Caspase-3 protein expression, in HT-29 cells, with a maximum obtained with the Lyc-Cub-NPs. These results indicate a proper inhibition of the PI3K/AKT/mTOR pathway and apoptosis induction. It has been well documented that an imbalance in the PI3K/AKT/mTOR pathway also exists in cancers, which confer resistance to apoptosis74,75,76,77,78. The findings are in line with those previously reported that activation of Akt suppresses proapoptotic proteins and favors the survival of cancerous cells, while inhibition of this pathway augments apoptotic signaling via Caspase-3 and other related executioners79,80,81,82. Higher effects elicited for Lyc-Cub-NPs indicate that cubic encapsulation enhances the lycopene ability to damp oncogenic signaling and trigger apoptosis more effectively than free lycopene alone and, thus, serve as a promising targeted therapeutic approach for colon cancer.
As far as we are aware, this is a pioneer study investigating the anticancer activity of lycopene cubosomal nanoparticles. Our findings support that cubsomes improve the bioavailability of lycopene and enhance the antioxidant, antitumor activities. Unlike traditional cytotoxic chemotherapies with low tumor specificity, targeted therapies exert direct antitumor effects by modulating oncogenic pathways of major relevance, inhibiting cell growth, inducing apoptosis, or promoting cellular differentiation. Targeted therapies may also indirectly inhibit the growth of tumors through the modulation of TME. For instance, targeted agents can alter vascularization and immune cell function in TME, thereby inhibiting tumor expansion and enhancing immune-mediated cytotoxicity and surveillance83.
Materials and methods
Drugs and chemicals
The capsules of 40 mg lycopene were supplied by Puritan’s Pride Inc. (Ronkonkoma, New York, USA). Glycerol α monooleate (TCI, Japan), polyvinyl alcohol (Merck, Germany), Poloxamer 407 (BASF Corporation, USA), deionized water (Waters, Milford, USA). All other reagents were of high analytical grade.
Preparation and characterization of Lyc-Cub-NPs
The melt dispersion emulsifying method was employed to manufacture lycopene cubsomes15,84. Poloxamer 407 (100 mg) was mixed with glycerol monooleate (400 mg). The dispersed phase of GMO and Poloxamer 407 was melted at 70 °C using a hot plate magnetic stirrer (Stuart, Caliber Scientific USA) to generate a homogeneous mixture. One lycopene capsules (equivalent to 40 mg) were accurately dissolved in the least amount of acetone and then uniformly added to the molten mixture. A preheated polyvinyl alcohol solution (10 ml) at 70 °C (2%) was added gradually to the previous molten mixture with continuous agitation for 10 min (400 rpm). The particle size of the colloidal dispersion was further adjusted with the aid of ultrasound treatment (Sonic Vibra Cell, USA). The colloid was incubated in an ice bath for 5 min (10 s pulse/10 s pause), 88% amplitude (130 W).
A spectrophotometric method (Shimadzu UV-VIS spectrophotometer, UV-1900I, Japan) was employed to calculate the lycopene content in the lycopene cubosome formulation85. A stock lycopene solution in n-hexane was prepared (1 mg/ml), a suitable dilution was conducted, and the standard lycopene was scanned from 200 to 600 nm (3 ml quartz cuvette/1 cm light travel length)86. Lycopene has three absorbance peaks, at 444, 471, and 503 nm87. The drug content was determined by calculating the lycopene concentration in the colloidal dispersion (the sample was diluted with n-hexane). Lycopene absorbance was measured at 471 nm, and lycopene concentration was computed. The test was carried out in triplicate. The particle size (PS), Zeta potential (Z), and polydispersity index (PDI) were determined (Zetasizer, Malvern Instruments). The samples were suitably diluted with deionized water, allowed to settle down for 5 min at ambient temperature before measurement88.
The drug content was estimated by transferring 200 µl of colloidal dispersion to a 10 ml volumetric flask, the volume was adjusted with the aid of n-hexane, the solution was suitably diluted with n-hexane, the absorbance was measured to calculate the total drug from the sample (measured concentration), and entrapment efficiency was calculated according to the following formula:
Dissolution test was performed in 500 ml dissolution medium of 0.5% (W/V) of Sodium lauryl sulphate (SLS) using USP baddle dissolution apparatus (COPLEY, England), at a speed of 100 rpm, and the temperature was adjusted at 37 ± 0.5 ᵒC89. Samples (3 ml) were collected at predetermined intervals (15, 30, 45, and 60 min) and filtered immediately through a 0.45 μm syringe filter before analysis. The dissolution medium volume was always constant (500 ml) due to the replacement of the removed sample with an equal volume of fresh dissolution medium (3 ml) after each sample collected. Lycopene concentration was estimated using a UV–Vis spectrophotometer at 471 nm. The test was performed in triplicate for Lyc-Cub-NPs. Dissolution efficiency was calculated according to the following equation89:
The characteristic honeycomb structure of a cubosome can be investigated by a transmission electron microscope (TEM) (JEM1400, JEOL Ltd., Tokyo, Japan). The colloidal dispersion was loaded onto a membrane-coated grid surface via filter paper; the samples were negatively stained with uranyl acetate dye for 3 min and dried at room temperature before being placed under the microscope. A scanning electron microscope (SEM) was utilized to study the surface characteristics of the cubosome (JEOL, JSM-6510LV, Japan). The colloidal dispersion was spread on a glass slide, after complete drying; the sample was transferred to the top of a cupper stub on a silicon electro-conductive chip. Before examination at different magnifications (20Kv electron acceleration), the sample was coated with gold. The FTIR is an effective tool for the identification of functional groups and the degree of interaction between active ingredients with other components. Lycopene and lycopene cubosome were tested by (FTIR; Perkin Elmer Fourier transform spectrometer, BRUKER, USA) and an air-cooled DTGs (deuterated triglycine sulfate) detector, and each sample was compressed with KBr into a disk using a hydraulic press. The disk was monitored using wavelengths ranging from 400 to 4000/cm.
Molecular docking
The docking target was the 3D structure of PI3Kα (PDB: 5XGI) (X). Lycopene’s structure was energy-minimized and docked into the ATP-binding pocket using SwissDock with the AutoDock Vina backend, following grid box parameters centered on known active site residues. The binding affinity of the top-scoring docking poses was analyzed59,90. PyMOL was employed to visualize protein-ligand interactions91, and PLIP was employed to generate schematic diagrams and characterize hydrophobic contacts91.
Antioxidant activity estimation
DPPH scavenging method
The DPPH radical scavenging experiment was conducted following the method reported by92 for Lycopene and Lyc-Cub-NPs at different doses. The reference chemical employed was ascorbic acid. The IC50 value was determined (n = 3) based on the concentration inhibition curve. The DPPH scavenging effect percentage was determined with the equation:
where A0 is the control response absorbance, and A1 is the absorbance with the addition of a test or reference sample.
ABTS scavenging method
With slight modification, ABTS radical scavenging activity was also determined for Lycopene and Lyc-Cub-NPs at various doses as per92. Absorbance at 734 nm was measured after incubation for six minutes with a UV/visible spectrophotometer (UV-VIS Milton Roy). The following equation was used to determine the levels of antioxidant activity:
A control = Negative control Absorption.
A sample = Six-minute sample absorbance.
Gallic acid was used as the standard chemical. A plot of the sample concentration needed to scavenge 50% of the free radicals of ABTS (n = 3) was used to determine the IC50 value.
Viability test of lycopene and Lyc-Cub-NPs against the HT-29 colon cancer cell line
Antitumor activity of Lycopene and Lyc-Cub-NPs was established in a viability assay against the HT-29 (ATCC® HTB-38™) cells, a human colorectal adenocarcinoma cell line, which were obtained from the American Type Culture Collection (ATCC) and cultured from the National Cancer Institute (NCI; Cairo, Egypt). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% Fetal Bovine Serum (Sigma-Aldrich, USA) and 1% penicillin-streptomycin (100 U/mL penicillin and 100 µg/mL streptomycin; Lonza, Switzerland). Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO₂. Cells were seeded at a density of 5 × 10⁴ cells per well in the medium. Lycopene and Lyc-Cub-NPs at doses of (0, 15.63, 31.25, 62.5, 125, and 250 µg/ml) were subsequently added to the cells following 48 h of culture in the exponential phase. 12 mM stock solution of MTT (10 µl) (Vybrant® MTT Cell Proliferation Assay Kit) was subsequently added to the wells. Subsequently, 50 µl of DMSO was added to each well following well mixing of the mixture with the pipette. It was incubated at 37 °C for 10 min. Absorbance was measured on a Bio-Tek Instruments Inc. (Santa Clara, CA, USA) ELx 800 microplate reader at 540 nm93. The following formula depicts the optical densities of treated cells (A) and the untreated cells (B):
In addition, IC50 was determined with the GraphPad Prism 10.2.3 software (San Diego, CA, USA).
Cell-cycle analysis
Flow cytometric evaluation of IC50 values yielded by the MTT assay (incubation time of 48 h) was employed to examine the cell-cycle distribution in the HT-29 Colon Cancer Cell line following treatment with Lycopene and Lyc-Cub-NPs. The medium culture was then withdrawn gradually after the cells were stimulated with Lycopene and Lyc-Cub-NPs. PBS was added and shaken gently before the removal of the PBS. 1 ml of trypsin was then added after introduction and well mixed, and left to digest in the incubator. The cells were taken from the incubator and transferred to a 3 ml tube in a bid to complete the trypsin digestion. The cells were resuspended and transferred into the centrifuge tube using a pipette. Centrifugation was used to discard the supernatant at room temperature for five minutes at 1000 rpm94. Three milliliters of PBS resuspension cells were added. Following their 75% alcohol resuscitation, the cells were kept overnight at 4 °C. Then, the supernatant was collected following 1000 rpm centrifugation for 5 min at room temperature. Following three PBS washes, the cell cycle was analyzed by flow cytometry (BD AccuriTM C6 Plus Flow Cytometer). The cells were stained in a propidium iodide staining solution for 30 min at 37C94. BD Biosciences AccuriTM C6 software approximated the percentage of cells in each cell-cycle phase.
Annexin V/PI double-staining
48 h after incubating, cells of the exponential growth phase were seeded into a well plate and treated with Lycopene and Lyc-Cub-NPs. After cells had been harvested, the culture medium was drawn into the centrifuge tube, 1 ml of 1× Binding Buffer was used to wash the cells, and 100 µl of 1× Binding Buffer was used to resuspend the cell pellet. The cells were spun down for 10 min at 300× g. Next, 10 µl of annexin-V, which is a fluorescent dye-labeled (annexin V-FITC), was added to every 106 cells. The cells were thoroughly mixed and incubated at room temperature for 15 min in the dark. Next, the 106 cells were each provided with 500 µl of 1× Binding Buffer for the second wash. Next, the cells were spun at 300× g for ten minutes. Then, 1× Binding Buffer (500 µl) was used to resuspend the cell pellet. Finally, before analysis by BD AccuriTM C6 Plus Flow Cytometer, 5 µl propidium iodide solution was added95.
Effect of lycopene and Lyc-Cub-NPs on PI3K, AKT, mTOR, caspase-3, and Bcl-2 gene expression in HT-29 colon cancer cells
The relative gene expression of PI3K, AKT, mTOR, Caspase-3, and Bcl-2 was determined by using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a housekeeping gene in qRT-PCR. The primer sequences are provided in Table 4. Total RNA was extracted utilizing TRIzol reagent (Catalog No. 15596026) obtained from Life Technologies, Thermo Fisher Scientific, located in Colorado Springs, USA. The reverse transcription process was carried out using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). The reaction mixes contained primers, complementary DNA amplicons, and Syber green master mix (Maxima SYBR Green/qPCR Master Mix, Thermo Fisher Scientific, Colorado Springs, USA). The gene expression was calculated using fold change techniques relative to the calibrator control (2−ΔΔCt)96.
Effect of lycopene and Lyc-Cub-NPs on PI3K, AKT, mTOR, caspase-3, and Bcl-2 markers in HT-29 colon cancer cells
The PI3K, Caspase-3, AKT, mTOR, and Bcl-2 levels in the HT-29 Colon Cancer Cell line following treatment with Lycopene and Lyc-Cub-NPs for 48 h were measured by ELISA kits from CUSABIO. Co, Houston, USA, AssayGenie. Co, Dublin, Ireland, and FineTest. Co, Hubei, China (Cat No. CSB-E08417h, CSB-E08856h, HUDL02301, HUFI01396, and EH0658, respectively). It was determined according to the kit manufacturer’s protocol.
Statistical analysis
Mean ± S.D. was shown for the data. Group differences were ascertained using one-way ANOVA followed by Tukey’s multiple comparisons. A p < 0.05 significance level was used to determine whether the differences seen were statistically significant or not. Multiple group statistical analysis was carried out using GraphPad Prism, version 9 (GraphPad Software Inc., La Jolla, CA, USA).
Data availability
The data are contained within the article.
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Acknowledgements
The authors extend their appreciation to Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2026R736), Princess Nourahbint Abdulrahman University, Riyadh, Saudi Arabia.
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Conceptualization, T.E.-M. and M.E.-N.; methodology, M.E.-N and E.E-Z.; validation, M.A.-S, T.E.-M, M.E.-B, M.E.-N., E.E-Z., H.K.B., M.S. and A. A.-R; formal analysis, M.A.-S, T.E.-M, M.E.-B, M.E.-N., E.E-Z., H.K.B., M.S. and A. A.-R; investigation, M.A.-S, T.E.-M, M.E.-B, M.E.-N., E.E-Z., H.K.B., M.S. and A. A.-R; resources, M.A.-S, T.E.-M, M.E.-B, M.E.-N., E.E-Z., H.K.B., M.S. and A. A.-R; data curation, M.E.-N., M.S. and E.E-Z.; writing—original draft preparation, M.E.-N., M.E.-B., M.S. and E.E-Z.; review and editing, M.A.-S, T.E.-M, M.E.-B, M.E.-N., E.E-Z., H.K.B., M.S. and A. A.-R. All authors have read and agreed to the published version of the manuscript.
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Alsunbul, M., El-Masry, T.A., El-Bouseary, M.M. et al. Cubosomal nanoparticles of lycopene as a novel platform for enhancement in antioxidant and anticancer properties with a molecular docking study. Sci Rep 16, 5941 (2026). https://doi.org/10.1038/s41598-026-36217-7
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DOI: https://doi.org/10.1038/s41598-026-36217-7
