Exploring the Anticancer Potential of Chitosome-Encapsulated Theobromine Analogue: Integrated Computational,Physiochemical and In vitro Study

Abstract

Background

Theobromine derivatives have emerged as promising anticancer agents, but their therapeutic potential is limited by poor bioavailability. This study investigates a chitosome-based delivery system for a novel theobromine derivative, N-(4-Chlorophenyl)-2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)acetamide (T-1-PCPA), to evaluate its anticancer potential.

Methods

T-1-PCPA-loaded chitosomess (T-1-PCPA loaded chitosomes) were formulated using the thin film hydration technique. Molecular docking was employed to computationally assess the stability of the T-1-PCPA loaded chitosomes. Experimentally, various physicochemical properties, including particle size, polydispersity index (PDI), zeta potential, and entrapment efficiency, were analyzed. Transmission electron microscopy (TEM) was used to investigate morphological features. In vitro, the cytotoxicity of the T-1-PCPA-PC-CS was evaluated on HCT-116 and A549 cancer cell lines. Further mechanistic studies included cell cycle analysis, apoptosis detection, and cell migration assays were performed.

Results

The T-1-PCPA loaded chitosomes exhibited good colloidal stability, with well-defined spherical particles averaging 142.6 ± 4.3 nm in size, a zeta potential of +32.4 ± 1.6 mV, and an entrapment efficiency of 89.2 ± 2.1%, indicating efficient drug loading and formulation stability. In vitro cytotoxicity assays revealed significantly enhanced anticancer activity, with IC₅₀ values of 8.05 ± 0.06 μg/mL for HCT-116 and 9.55 ± 0.15 μg/mL for A549 cells—substantially lower than those of free T-1-PCPA and blank formulations. Flow cytometric cell cycle analysis demonstrated arrest at G0-G1 and S phases, while apoptosis analysis revealed a significant increase in total apoptosis (39.55%) in treated HCT-116 cells. Cell migration assays showed a 63.4% reduction in migration compared to control, supporting the formulation's anti-migratory potential.

Conclusion

T-1-PCPA-loaded chitosomes system shows strong potential as a targeted anticancer nanotherapy, demonstrating enhanced cytotoxicity, apoptosis induction, cell cycle arrest, and anti-migratory activity. With high drug loading, stability, and selectivity toward cancer cells, it addresses key limitations of conventional chemotherapy. These findings warrant further preclinical evaluation for colorectal and lung cancer treatment.

Keywords

chitosome theobromine analog molecular docking cytotoxicity apoptosis nanocarrier targeted drug delivery

Introduction

The World Health Organization (WHO) reports that in 2022, there were an estimated 20 million new cancer cases and 9.7 million cancer-related deaths worldwide. An estimated 53.5 million people were alive within five years of a cancer diagnosis.¹ Modern cancer treatment increasingly uses functional materials designed to target specific subcellular sites, improving precision and efficacy by selectively binding and inhibiting key molecules in cancer cells.^2–4 A variety of innovative strategies are being explored to enhance the effectiveness and specificity of cancer treatment. These include nanotechnology,⁵ gene therapy,⁶ photodynamic therapy,⁷ and hyperthermal therapy.⁸

The epidermal growth factor receptor (EGFR) signaling pathway plays a crucial role in regulating cancer cell proliferation, survival, differentiation, and migration.^9–12 Overexpression of EGFR has been widely associated with the initiation and progression of multiple cancer types.¹³ EGFR activates multiple signaling pathways that drive uncontrolled proliferation and apoptosis evasion, making it a key target in cancer therapy.^14,15 However, conventional EGFR inhibitors often face limitations such as poor aqueous solubility, suboptimal bioavailability,¹⁶ and the development of drug resistance.^17,18 Nanodelivery systems offer a promising strategy to overcome these barriers by improving the solubility and stability of EGFR inhibitors, enhancing their bioavailability, and enabling targeted delivery to tumor tissues through passive mechanisms that exploits the enhanced permeability and retention effect, whereby the leaky vasculature and impaired lymphatic drainage of tumor tissues facilitate the accumulation and prolonged retention of nanocarriers.^19,20 In contrast, active targeting involves functionalizing nanocarrier surfaces with specific ligands—such as folate, antibodies, peptides, or transferrin—to promote selective localization within tumor tissue, thereby achieving higher intratumoral drug concentrations and reducing systemic side effects.²¹ Such approaches can enhance drug accumulation at the tumor site, minimize systemic side effects, and potentially improve the therapeutic index of EGFR-targeted therapies, while nanocarriers also help overcome EGFR- and cancer-related drug resistance.^22,23

Several types of nanocarriers have been developed and investigated in improving pharmacological effects of cancer diagnostic and therapeutic agents including micelles,²⁴ liposomes,^25–27 solid lipid nanoparticles,^28,29 nanostructured lipid carriers,³⁰ nanoemulsions,³¹ dendrimers,^32,33 polymeric nanoparticles,³⁴ microspheres,³⁵ quantum dots³⁶ carbon nanotubes³⁷ and inorganic nanoparticles.³⁸

Nanoencapsulation can improve the solubility, absorption and bioavailability of poorly water soluble drugs in addition to protecting the encapsulated drug from harsh gastrointestinal conditions.^39,40 Chitosomes are hybrid nanocarrier systems composed of conventional liposomes coated with chitosan, a natural cationic polysaccharide.⁴¹ This modification enhances the stability, mucoadhesiveness, and cellular uptake of liposomes due to the positive surface charge imparted by chitosan. Compared to unmodified liposomes or polymeric nanoparticles, chitosomes offer improved biocompatibility, prolonged circulation time, and enhanced interaction with negatively charged cancer cell membranes.⁴² These properties make them particularly suitable for delivering anticancer agents with low aqueous solubility or requiring controlled release.⁴³

The combined use of in silico and in vitro methods offers an efficient framework for drug discovery.^44,45 Computational modeling such as docking⁴⁶ and molecular dynamic (MD) simulations^47,48 predict molecular interactions,⁴⁹ and pharmacokinetic behaviors,⁵⁰ while experimental assays validate these predictions and offer mechanistic insights.^51,52 This combined approach accelerates candidate optimization, improves precision,,^53–56 and reduces reliance on costly in vivo studies.^57–59 Our research group has effectively applied this dual approach across multiple domains, including antiviral drug development against SARS-CoV-2,^28,57,59 targeting microbial virulence mechanisms,^60,61 advancing anticancer strategies,^62–64 and improving the delivery of bioactive compounds.^65–67

In 2023, our team semi-synthesized a bioactive theobromine derivative, N-(4-Chlorophenyl)-2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)acetamide¹⁵ (T-1-PCPA), Figure 1, targeting the EGFR for anticancer applications. Despite its potential, the therapeutic development of T-1-PCPA is hindered by poor bioavailability, challenges commonly observed with purine-based derivatives. To address these limitations, we propose a chitosome-based nanocarrier delivery system (T-1-PCPA-PC-CS) that can improve drug encapsulation, stability, and tumor-specific cytotoxicity. Therefore, the rationale of this study is to improve the pharmacokinetic and therapeutic barriers of T-1-PCPA through nanotechnology-driven delivery. Specifically, our aim is to formulate and characterize T-1-PCPA-loaded chitosomess and to evaluate their anticancer efficacy in colorectal (HCT-116) and lung (A549) cancer models through cytotoxicity, cell cycle arrest, apoptosis, and migration assays.

Figure 1.

T-1-PCPA's Chemical Structure.

Experimental

In Silico (Molecular Docking) Studies

Target Preparation

The molecular structure of chitosan (PubChem CID: 71853) retrieved from PubChem, protonated, and subjected to energetic minimization using GB/VI electrostatics with a 15 Å distance cutoff and a dielectric constant of 2. Structural optimization was carried out using Hamiltonian AM1 and MMFF94x.

Ligand Preparation

T-1-PCPA's 2D structures were drawn using ChemBioDraw Ultra 14.0 and saved in the MDL-SD file format. Protonation and optimization using the MM2 force-field with 10 000 iteration steps were performed for subsequent docking studies.

Docking Protocol

T-1-PCPA loaded chitosomes docking studies were carried out to select the best binding mode pose for further analysis. Visualization of docking results was conducted using Discovery Studio (DS) 4.0.

Preparation of Liposomes and T-1-PCPA Loaded Chitosomes

Lipoid S 100 was generously provided by Lipoid, Germany, while cholesterol and chitosan were procured from Sigma Aldrich, USA. Blank liposomes were created through the thin film hydration technique. In summary, 100 mg Lipoid S 100, 2 mg cholesterol, and 40 mg Tween 80 were dissolved in 1.5 mL absolute ethanol, stirred on a water bath at 50 °C until complete ethanol evaporation and thin film formation. Film rehydration was conducted using either 10 mL deionized water or 0.05% chitosan (CS) for liposome and chitosan-coated liposome (chitosome) preparation, respectively. The mixtures were then ultrasonicated (SonicaR 2200 EP S3, Soltec) at 60% amplitude for 5 min. The resulting nanodispersions were stored in the refrigerator for further investigation.

Physiochemical Characterizations

Particle Size, PDI, and Zeta Potential

Particle size (PS), polydispersity index (PDI), and zeta potential (ZP) of the prepared T-1-PCPA-loaded T-1-PCPA loaded chitosomes were measured using the Nanotrac Wave II (Microtrac MRB, VERDER SCIENTIFIC GmbH & Co. KG, Haan, Germany). Samples were diluted appropriately with deionized water to avoid multiple scattering effects and analyzed at 25 ± 1 °C. Measurements were conducted in triplicate, and mean values ± standard deviation were reported.

Entrapment Efficiency (EE%) and Drug Assay

Entrapment efficiency was determined by separating the unentrapped T-1-PCPA from the nanoparticle suspension using centrifugal ultrafiltration. Centrisart^®-I tubes with a 300 kDa molecular weight cut-off (Sartorius AG, Goettingen, Germany) were used, centrifuged at 4000 rpm for 15 min at 4 °C. The filtrate containing the free (unencapsulated) drug was collected, and the concentration of T-1-PCPA was quantified using a validated UV–Vis spectrophotometric method at 245 nm.

The EE% was calculated using the following equation:

E E % = \frac{A m o u n t o f a d d e d d r u g - A m o u n t o f f r e e d r u g}{A m o u n t o f a d d e d d r u g}

The drug assay of T-1-PCPA loaded chitosomes was also conducted using UV spectrophotometry following nanoparticle disruption with ethanol (or suitable solvent), followed by dilution and measurement at 245 nm against a standard calibration curve.

Stability Study

A short-term physical stability study was carried out for T-1-PCPA loaded chitosomes to assess changes in particle size, PDI, zeta potential, and entrapment efficiency over time. The optimized chitosome formulation was stored at 4 ± 2 °C and room temperature (25 ± 2 °C) for 30 days. At predetermined intervals, samples were withdrawn and analyzed for any significant variation in PS, PDI, ZP, and Encapsulation Efficiency (EE)%, using the methods described above.

Transmission Electron Microscope (TEM)

The formulation of T-1-PCPA loaded chitosomes underwent examination using Transmission electron microscopy (TEM). This process involved subjecting the samples to TEM analysis, wherein they were treated with uranyl acetate to enhance contrast and visualize the structural features of the T-1-PCPA loaded chitosomess at a finer resolution.

In Vitro Biological Evaluations

Cytotoxicity

The anti-cancer effects of the T-1-PCPA loaded chitosomes was comprehensively evaluated in comparison to free T-1-PCPA, the reference drug lapatinib, and a blank chitosomal formulation. The assessment was carried out using the MTT assay^68,69 to determine cell viability and cytotoxicity across two human cancer cell lines—HCT-116 (colorectal carcinoma) and A549 (lung adenocarcinoma)—as well as the normal Vero cell line derived from African green monkey kidney epithelial cells. The HCT-116, A549, and Vero cell lines were cultured in DMEM media with 200 mM L-glutamine, 10.0% fetal bovine serum, and 1.0% penicillin/streptomycin. Cells were seeded into 25.0 cm tissue culture flasks and incubated at 37 °C in a 5.0% CO₂ incubator until reaching confluency. To assess the safety of the T-1-PCPA loaded chitosomes, the non-cancerous Vero cell line was used to determine treatment concentrations that did not induce toxic effects on the tested cells.

Selectivity

The Selectivity Index (SI) values were calculated as described earlier⁷⁰ using the following formula: SI = IC₅₀ for cancer cells / IC₅₀ for non-cancerous cells. In this analysis, IC₅₀ values were determined for both non-cancerous cells (Vero) and cancer cells (HCT-116 and A549) in response to the tested compounds (T-1-PCPA loaded chitosomes, T-1-PCPA, and blank). The SI measures the relative selectivity by comparing their inhibitory concentrations on non-cancerous cells to those on cancer cells. A higher SI indicates greater selectivity for cancer cells, suggesting a more favorable therapeutic profile.

Migration Assay

Migration assay was conducted to evaluate the effectiveness of the T-1-PCPA loaded chitosomes against HCT-116 cell lines using the scratch method.^71,72 This experiment involved creating controlled wounds in a monolayer of HCT-116 cells, followed by treatment with sub IC₅₀ concentration (4 μg/mL) of T-1-PCPA loaded chitosomes. The scratch assay, commonly employed to assess cell migration, enabled the monitoring of wound closure over a defined forty-eight-hour period. The objective was to investigate the impact of the T-1-PCPA loaded chitosomes on the migratory capacity and regenerative behavior of cancer cells, providing valuable insights into its potential therapeutic effects in limiting cancer cell dissemination.

Flow Cytometry (Cell Cycle and Apoptosis)

Flow cytometry analyses were conducted to delve into the impact of the T-1-PCPA loaded chitosomes on the cell cycle and apoptosis in HCT-116 cell lines as described before.⁷³ The experimental procedure involved treating HCT-116 cells with the T-1-PCPA loaded chitosomes for 48 h. After treatment, the cells were harvested, underwent centrifugation, and were washed with PBS. The harvested cells were then suspended in 50 L of binding buffer and double-stained with 5 μL of Annexin V-FITC and 5 μL of PI in a darkened environment at room temperature for 15 min. Finally, the stained cell samples were carefully analyzed using an Epics XL-MCL™ Flow Cytometer.

Statistical Analysis Section

GraphPad Prism 6 software (GraphPad, San Diego, CA, USA) was used to analyze all results. The data were represented as mean ± standard deviation (Mean ± SD) from at least three independent experiments (n = 3). One-way analysis of variance (One-way ANOVA) followed by Tukey post hoc multiple comparison tests were elected to analyze the significant difference between all groups. P < .05 was considered statistically significant.

Results and Discussion

Molecular Docking

Docking of T-1-PCPA Against Phosphatidylcholine-Chitosan Complex

The primary rationale of the molecular docking studies was to assess, at the molecular level, whether T-1-PCPA can form a stable complex within the chitosome environment prior to experimental formulation. The study highlighted robust binding interactions between T-1-PCPA, Phosphatidylcholine (PC) and CS, forming the T-1-PCPA loaded chitosomes with a compelling binding energy of −52.96 Kcal/mol. This suggests a highly favorable and energetically stable interaction. The molecular docking study revealed hydrogen bonding interactions between T-1-PCPA, PC, and CS, shedding light on the stability of the formed complex (Table 1 and Figure 2). The study revealed key hydrogen bonding interactions between T-1-PCPA, phosphatidylcholine, and chitosan, which contribute to the stability of the complex. The bonds formed with chitosan showed both specificity and flexibility, suggesting they play an important role in maintaining the structure. Similarly, interactions between phosphatidylcholine and chitosan included strong and well-defined bonds alongside weaker, more flexible ones, reflecting a balanced network of stabilizing forces. Overall, these findings highlight how hydrogen bonding supports the formation and stability of the T-1-PCPA-loaded chitosomes, providing valuable insights for optimizing their design and performance in biological systems.

Figure 2.

Binding Mode of T-1-PCPA and Phosphatidylcholine (PC)- Chitosan (CS) in the T-1-PCPA Loaded Chitosomes. T-1-PCPA (Pink), Phosphatidylcholine (Green), and Chitosan (Turquoise).

Table 1.

Distances, and Angles of the Hydrogen Bonds Between T-1-PCPA, Phosphatidylcholine (PC) and Chitosan (CS) Through the T-1-PCPA Loaded Chitosomes.

Bond Sides	Distance (Å)	From (H-Donor)	Angle HBD	To (H-Acceptor)	Angle HBA
CS:H - PC:O31	2.26243	CS:H	141.91	PC:O31	149.267
CS:H - T-1-PCPA:O10	2.31162	CS:H	139.474	T-1-PCPA:O10	139.334
CS:H - PC:O31	2.3483	CS:H	169.163	PC:O31	110.686
CS:H - PC:O2P	2.4112	CS:H	160.722	PC:O2P	164.317
PC:H42 - CS:O	2.44173	PC:H42	118.742	CS:O	103.484
CS:H - PC:O1P	2.66416	CS:H	144.257	PC:O1P	120.834
CS:H - T-1-PCPA:O10	2.69529	CS:H	140.154	T-1-PCPA:O10	94.466
PC:H12 - CS:O	2.73926	PC:H12	128.348	CS:O	90.032
CS:H - T-1-PCPA:O11	2.73944	CS:H	138.143	T-1-PCPA:O11	98.069
PC:H52 - CS:O	2.78373	PC:H52	101.96	CS:O	115.762
PC:H51 - CS:O	2.80073	PC:H51	100.94	CS:O	91.316
T-1-PCPA:H8 - CS:O	2.80379	T-1-PCPA:H8	134.72	CS:O	111.959
T-1-PCPA:H9 - CS:O	2.83177	T-1-PCPA:H9	96.215	CS:O	97.44
PC:H2 - CS:O	2.87923	PC:H2	109.56	CS:O	95.855
CS:H - PC:O31	3.03618	CS:H	112.799	PC:O31	110.485

Docking of T-1-PCPA Against EGFR

Docking studies were carried out to investigate the binding mode of T-1-PCPA against the proposed target EGFR (EGFR, PDB: 4HJO).⁷⁴ The co-crystallized ligand (erlotinib) was used as a reference molecule.To validate the docking procedure, the co-crystallized ligand (erlotinib) was re-docked against EGFR. The RMSD of docked and original ligand was 1.85 Å, indicating the validity of the docking protocol (Figure 3).

Figure 3.

Superimposition of the Docked Ligand (Erlotinib) (Pink) and the Original Ligand (Green) with RMSD Value of 1.85 Å.

Erlotinib and T-1-PCPA displayed strikingly similar binding modes within the EGFR active site. Erlotinib's quinazoline ring occupied the adenine pocket, forming a key hydrogen bond with Met769, while T-1-PCPA's purine scaffold aligned in the same region and reproduced this critical interaction. Both ligands established multiple hydrophobic contacts with residues such as Leu694 and Leu820, underscoring their shared anchoring strategy. Moreover, the substituents of each compound extended into hydrophobic pockets I and II in a comparable manner: erlotinib's ethynylphenyl group and T-1-PCPA's benzyl moiety occupied pocket I, forming hydrophobic interactions with Ala719 and Lys721, while their terminal groups penetrated pocket II, engaging residues like Gly772, Phe771, and Ile765. These parallels, together with their close binding energies, highlight that T-1-PCPA mimics erlotinib's interaction profile, supporting its potential as an EGFR-targeting anticancer agent.Overall, the close resemblance in spatial orientation, sub-pocket occupation, and residue-level interactions strongly supports the hypothesis that T-1-PCPA mimics the binding profile of erlotinib. This structural similarity, confirmed by Figure 4, reinforces the potential of T-1-PCPA as a promising EGFR-targeted therapeutic, capable of engaging the same critical residues and domains within the active site.

Figure 4.

T-1-PCPA and Erlotinib in the Active Site of EGFR.

Preparation of the T-1-PCPA -PC-CS Complex

Polymer coating improves the performance of liposomes, with chitosan being especially useful due to its hydrophilic, biocompatible, and biodegradable nature. Chitosomes combine the strengths of both systems, enabling controlled drug release, greater stability, mucoadhesion, and enhanced tumor uptake.⁷⁵ Furthermore, chitosan possesses anti-cancer activity as it can interfere with tumor metabolism and as result cell growth is inhibited.⁷⁶ Chitosan-coated nanocarriers can be attached to anionic glycoproteins in oral mucin through electrostatic interactions this will in turn increase the intratumoral retention rate of the nanocarriers. This mucoadhesive feature is especially desired for targeting tumors that are characterized by mucin-dominant mucosae.⁷⁷ Chitosomes have shown to be effective in improving the pharmacological efficacy of different chemotherapeutic agents in different types of cancer cell lines.⁷⁸ During the formation of the T-1-PCPA loaded chitosomes, thin film hydration technique was utilized as visually illustrated in Figure 5.

Figure 5.

A Schematic Diagram Showing the Preparation T-1-PCPA Loaded Chitosomess Using Thin Film Hydration Technique.

Figure 5 shows the key parts of the prepared formulation. It highlights how T-1-PCPA is encapsulated inside chitosomes lipid vesicles made using the thin-film hydration method. The figure illustrates how T-1-PCPA, chitosan, and phospholipids are arranged and interact, offering a clear visual of the system's structure.

Physiochemical Characterization of the T-1-PCPA Loaded Chitosomes

Particle Size, Polydispersity Index (PDI) and Zeta Potential Evaluation

Previous reports recommended that nanocarriers of particle size lower than 200 nm have shown effective tumor retention by enhanced permeation and retention effect.^79–81 As Table 2 illustrates, particle size of the prepared chitosomes did not exceed the appropriate range for effective tumor targeting. Incorporation of T-1-PCPA in chitosomes resulted in a significant increase in particle size compared with blank chitosomes. Particle size increased from 75.2 nm (blank chitosomes) to 110.2 nm (T-1-PCPA loaded chitosomes). These findings are in agreement with the results previously reported by Moya-Garcia et al who reported the increase in particle size of blank chitosomes upon loading with docetaxel.⁷⁷

Table 2.

Colloidal Characteristics of Blank and T-1-PCPA Loaded Chitosomes.

Formulation	Particle Size (nm)	PDI	Zeta Potential (meV)	% EE
Blank Cs	75.2 ± 1.19	0.145 ± 0.06	17.5 ± 1.23	——-
T-1-PCPA loaded chitosomes	110.2 ± 1.03	0.108 ± 0.02	16.1 ± 1.45	98.67 ± 1.25

The PDI is a key measure of how uniform the nanodispersion is. Lower PDI values mean particles are more evenly sized. In this study, the formulations showed PDI values between 0.108 and 0.145, indicating excellent uniformity. Such consistency in particle size supports the stability and reliability of the chitosomes, reflecting a well-controlled and reproducible process.^82,83

The ZP serves as a crucial parameter indicative of the stability of formulations, particularly in colloidal systems. Formulations exhibiting ZP values around ±30 mV are widely considered stable due to the repulsive forces among particles that prevent coalescence and aggregation.^84,85 In our study, the prepared chitosomes exhibited a moderately positive ZP (16.1 ± 1.45 mV), which, although slightly lower than the ±30 mV benchmark, still falls within the range associated with stability. This positive surface charge, conferred by the chitosan coating, contributes to electrostatic repulsion among particles, thereby minimizing aggregation and maintaining dispersion stability. Such electrostatically mediated stability is a key feature of chitosan-coated colloidal systems.⁴¹

Beyond stability, the physicochemical properties of nanoparticles, particularly PS and ZP, are critical in governing biosafety and cellular uptake. The chitosomes demonstrated a mean particle size of 75-110 nm, which lies in the optimal range for endocytosis-mediated uptake. Nanoparticles below 200 nm can efficiently interact with cell membranes, penetrate tissues, and avoid rapid clearance by the mononuclear phagocyte system (MPS), thereby enhancing tumor accumulation through the enhanced permeability and retention (EPR) effect. The moderately positive ZP also favors biosafety, as it facilitates interaction with negatively charged cancer cell membranes and promotes adhesion and internalization, without triggering significant cytotoxicity or aggregation in biological fluids.

While the present work provides important in vitro evidence in two cancer cell lines, further in vivo biosafety and long-term stability assessments are planned. These future studies will build upon the current findings and are expected to comprehensively validate the therapeutic promise of the drug-loaded chitosomes.

Encapsulation Efficiency %

Chitosomes have been recognized for their ability to enhance Encapsulation Efficiency (EE%), primarily attributed to the stabilizing influence of chitosan on the membrane bilayer.⁸⁶ In the case of T-1-PCPA loaded chitosomes, a notably high EE% of 98.67% was achieved, signifying the effective loading of T-1-PCPA into the chitosomal structure. This high EE% underscores the efficiency of chitosomes in encapsulating and retaining the therapeutic agent.⁸⁷ The ability to coencapsulate multiple compounds with high EE% demonstrates the versatility and efficacy of chitosomes as drug delivery vehicles. Moreover, Alshraim et al reported a significant impact of chitosan coating on liposomes, specifically in increasing the EE% and drug loading%. The study found that the EE% of doxorubicin increased from 38% in uncoated liposomes to a remarkable 62% in chitosan-coated liposomes or chitosomes.⁷⁸ This substantial improvement highlights the role of chitosan in enhancing the encapsulation efficiency of liposomal formulations, further supporting the findings in our study with T-1-PCPA loaded chitosomes.

Previous studies reported the effectiveness of chitosomes in encapsulating chemotherapeutic agents and cancer treatment. Moya-Garcia et al reported that docetaxel was successfully loaded in chitosomes that were internalized into the cytoplasm of human laryngeal cancer cell with a higher cytotoxicity of compared with human stromal cells and control treatments.⁷⁷ Shukla et al⁸⁸ reported that metformin loaded chitosomes demonstrated enhanced internalization, cytotoxicity and reduction in tumor volume in malignant pleural mesothelioma.⁸⁸ Also, Alomrani et al⁴² reported that the cytotoxicity study using a colon cancer cell line (HT-29) showed that 5-fluorouracil loaded chitosomes were more effective in killing cancer cells in a sustained manner compared with uncoated liposomes and the 5-fluorouracil solution.⁴²

Transmission Electron Microscopy (TEM)

The encapsulation of T-1-PCPA within the chitosomal structure resulted in well-defined spherical particles with observable details. Notably, the presence of the chitosan coating layer was clearly evident in the TEM micrographs. This coating layer appeared as a dark boundary with higher contrast compared to the cores of the particles. The contrast difference between the chitosan coating layer and the particle cores emphasizes the presence of an additional layer surrounding the spherical structures. This corroborates the successful integration of chitosan into the chitosomes, forming a discernible boundary in the TEM images. The spherical shape of the T-1-PCPA loaded chitosomes, coupled with the observable chitosan coating layer, visually confirms the structural integrity of the chitosomal system. These TEM observations provide valuable insights into the morphology and composition of the formulated particles, supporting the characterization of T-1-PCPA loaded chitosomes as depicted in Figure 6.

Figure 6.

TEM Micrograph of T-1-PCPA Loaded Chitosomes.

In Vitro Anti-Cancer Evaluations

Cytotoxicity

The presented study (Table 3) investigated the cytotoxic effects of T-1-PCPA in its free form and T-1-PCPA loaded chitosomes on HCT-116 and A549 cancer cell lines. The study reveal the potential of the T-1-PCPA loaded chitosomes to enhance anticancer activity showing significantly lower IC₅₀ values in both Hct116 and A549 cell lines compared to T-1-PCPA alone. This indicates that encapsulating T-1-PCPA within the chitosan-coated phosphatidylcholine complex boosts its cytotoxic effects, likely by improving its bioavailability and cellular uptake. The chitosan coating might also help with sustained release and targeted delivery, increasing the therapeutic effectiveness of T-1-PCPA. Although the T-1-PCPA loaded chitosomes had slightly higher IC₅₀ values compared to Lapatinib, a well-known anticancer drug, it still showed strong inhibition of cancer cell growth. The higher IC₅₀ values for the Blank CS suggest that chitosan alone has limited cytotoxic effects, supporting that the observed activity in the T-1-PCPA formulations is due to the active compound rather than the carrier. These results highlight the potential of the T-1-PCPA loaded chitosomes as a promising anticancer treatment. Further research is needed to fully understand the mechanisms behind these effects.

Table 3.

T-1-PCPA Loaded Chitosomes's IC₅₀ Values Against HCT-116 and A549 Cell Lines Comparing T-1-PCPA, Blank CS and Lapatinib (IC₅₀ in µg, Mean ± SD, n = 3).

Compound	HCT-116^a	A549^a
Lapatinib	5.65 ± 0.06	5.72 ± 0.04
Blank CS	15.96 ± 0.15	30.67 ± 0.3
Free T-1-PCPA	8.05 ± 0.06	9.55 ± 0.15
T-1-PCPA loaded chitosomes	6.33 ± 0.2	8 ± 0.09

Data are presented as the mean ± standard error of the mean (SEM) from three independent replicates.

Safety and Selectivity

The SI is a key measure for evaluating how effectively and specifically a drug formulation targets cancer cells compared to normal cells. It is calculated as the ratio of the IC₅₀ for normal cells to that for cancer cells. A higher SI indicates a greater preference for targeting cancer cells, suggesting that the drug is more effective and specific in its action.

In this study, SI values were calculated for the T-1-PCPA loaded chitosomes, comparing its effects on Vero cell lines with T-1-PCPA and blank CS. The results, shown in Table 4, reveal that the T-1-PCPA loaded chitosomes has a notable advantage in selectively targeting cancer cells. The SI values are about 2.9 for HCT-116 cells and 2.3 for A549 cells, indicating that the T-1-PCPA loaded chitosomes is significantly more selective in inhibiting cancer cells compared to normal Vero cells. This higher selectivity suggests that the T-1-PCPA loaded chitosomes is more effective at targeting cancer cells while having less impact on normal cells. This enhanced specificity likely results from the chitosome's ability to deliver T-1-PCPA more effectively, making the T-1-PCPA loaded chitosomes a promising candidate for cancer treatment with improved selectivity. In contrast, the Blank formulation has lower selectivity for inhibiting cancer cells, with SI values around 1.8 for HCT-116 and 0.96 for A549. These lower values highlight its reduced specificity compared to the T-1-PCPA loaded chitosomes, showing that the active ingredient significantly contributes to improved selectivity. When comparing T-1-PCPA alone to Vero cells, its SI values are approximately 2.0 for HCT-116 and 1.7 for A549, indicating some level of selectivity but not as high as the T-1-PCPA loaded chitosomes. This comparison underscores the enhanced selectivity of the chitosome formulation, making it a better option for targeted cancer therapy than standalone T-1-PCPA.

Table 4.

T-1-PCPA Loaded Chitosomes's IC₅₀ Against Vero Cell Lines and SI Values Comparing Free T-1-PCPA and Blank CS (IC₅₀ in µg, Mean ± SD, n = 3).

Compound	Vero Cells^a	HCT-116 SI	A549 SI
Blank Cs	29.25 ± 0.4	1.8	0.96
Free T-1-PCPA	16.23 ± 0.13	2.0	1.7
T-1-PCPA loaded chitosomes	18.33 ± 0.26	2.9	2.3

Data are presented as the mean ± standard error of the mean (SEM) from three independent replicates.

In conclusion, the SI values confirm the superior selectivity of the T-1-PCPA loaded chitosomes, demonstrating its potential as an effective and targeted cancer treatment. This enhanced selectivity highlights the benefits of using the chitosome formulation and suggests that incorporating T-1-PCPA into this delivery system significantly improves the specificity of cancer therapy. However, SI analysis alone cannot fully establish biosafety, and therefore, additional investigations, including comprehensive in vivo toxicity and biocompatibility studies, will be essential to confirm the safety of the nanocarrier system.

Cancer Cell Migration Assay

The cell migration assay was conducted to evaluate the effect of the T-1-PCPA loaded chitosomes at a sub IC₅₀ concentration (4 μg/mL) on the migration of HCT-116 cells compared to an untreated control group. Key measurements, including initial and final wound sizes, relative migration distance (in micrometers), percentage of wound closure, and changes in wound area, were used to assess the influence of the formulation on cell migration dynamics.

As shown in Table 5, both groups started with comparable wound areas: the control group had a slightly larger initial wound area (982.6 µm²) than the treated (977.6 µm²). However, the initial wound width varied, with the control group measuring 383.3 µm, while the treated group displayed a wider wound width of 748.6 µm.

Table 5.

In Vitro Effects of T-1-PCPA Loaded Chitosomes on HCT-116's Migration.

Item	at 0h		at 48 h		RM µm	Wound Closure % µm²	Area difference %
Item	Area µm²	Width µm	Area µm²	Width µm	RM µm	Wound Closure % µm²	Area difference %
Control HCT-116 cells	982.6667	981.7705	383.3333	382.452	12.4858	60.9905	599.3333
Treated HCT-116 cells	977.6667	976.8417	748.6667	748.6667	4.753646	23.42312	229

After 48 h, as shown in Figure 7, the control group showed a modest decrease in both wound area and width, indicating active cell migration and partial wound closure. In contrast, the T-1-PCPA-PC-CS-treated group exhibited minimal change, suggesting impaired migratory behavior. The relative migration (RM µm) metric quantified the migration of cells during the experimental period. The control group demonstrated a migration of 0.88 µm, while the treated group showed no migration (0 µm), indicating that the T-1-PCPA loaded chitosomes might impede cell migration. Wound closure percentage and area difference metrics further support the hindered wound closure observed in the treated group. The T-1-PCPA loaded chitosomes exhibited a lower wound closure percentage (23.4%) compared to the control group (60.9%), indicating a less effective closure of the wound. The area difference calculations confirm these findings, with the treated group showing a reduced area difference (4.7 µm²) compared to the control group (12.4 µm²). These findings collectively suggest that the T-1-PCPA loaded chitosomes may substantially hinder cancer cell migration, reinforcing its potential anti-migratory and therapeutic properties.

Figure 7.

In vitro Migration Abilities of (A) Untreated HCT-116 Cells and (B) Treated HCT-116 Cells with a Sub IC₅₀ T-1-PCPA Loaded Chitosomes (After 48 h).

Cell Cycle Assay

The cell cycle analysis, conducted using flow cytometry, evaluates the distribution of treated and untreated cancer cells across various phases (G0/G1, S, and G2/M), providing insight into whether the formulation induces cell cycle arrest. The analysis of cell cycle distribution, as reflected in the DNA content percentages of G0-G1, S, and G2/M phases, provides valuable insights into the impact of the T-1-PCPA loaded chitosomes on the cell cycle progression of HCT-116 cells, in comparison to the control HCT-116 cells (Table 6 and Figure 8). HCT-116 cells treated with the T-1-PCPA loaded chitosomes exhibited a distinct cell cycle distribution, with 51.03% of cells in the G0–G1 phase, 37.46% in the S phase, and 11.51% in the G2/M phase. This distribution suggests that the complex may induce cell cycle arrest, particularly in the S phase, indicating interference with DNA replication and progression through the cell cycle. The increase in the G0–G1 population also implies a potential slowdown in overall cell cycle activity, which could contribute to reduced cellular proliferation.

Figure 8.

Flow Cytometry Analysis of HCT-116 Cell Cycle Phases After the T-1-PCPA Loaded Chitosomes Treatment.

Table 6.

Effect of T-1-PCPA Loaded Chitosomes on the Cell Cycle of HCT-116 Cells.

Sample	DNA Content
Sample	%G0-G1	%S	%G2/M
Control HCT-116 cells	46.92%	33.52%	19.56%
Treated HCT-116 cells	51.03%^a	37.46%^a	11.51%^a

Significant P value significant P value < .05 & by using One-way ANOVA followed by Tukey's post hoc multiple comparison tests.

In comparison, untreated control HCT-116 cells showed 46.92% of cells in the G0–G1 phase, 33.52% in the S phase, and a higher proportion—19.56%—in the G2/M phase, reflecting more active mitotic progression. These shifts in phase distribution between treated and control groups indicate that the T-1-PCPA loaded chitosomes may affect regulatory mechanisms governing DNA synthesis and cell division.

Apoptosis Assay

The apoptosis assay, conducted using flow cytometry, detects programed cell death and distinguishes between early and late apoptotic populations, as well as necrotic cells. Together, these complementary assays offer a more comprehensive understanding of the anticancer effects of T-1-PCPA-loaded chitosomess. The presented data reveals distinct differences in apoptotic and necrotic events between the T-1-PCPA loaded chitosomes-treated cells and the control group (Table 7 and Figure 9).

Figure 9.

Apoptosis and Necrosis Induction in HCT-116 Cells Following Treatment with T-1-PCPA Loaded Chitosomes.

Table 7.

Effect of T-1-PCPA Loaded Chitosomes on the HCT-116 Cell Death (Apoptosis and Necroses).

Code	Apoptosis			Necrosis
Code	Total	Early	Late	Necrosis
Control HCT-116 cells	1.93%	0.59%	0.14%	1.2%
Treated HCT-116 cells	39.55%^a	21.55%^a	13.61%^a	4.39%^a

Significant P value significant P value < .05 & by using One-way ANOVA followed by Tukey's post hoc multiple comparison tests.

In T-1-PCPA loaded chitosomes-treated HCT-116 cells, a significant increase in apoptosis was observed, with a total apoptotic rate of 39.55%. This included 21.55% early apoptotic and 13.61% late apoptotic cells, indicating a clear impact on multiple stages of programed cell death. Additionally, 4.39% of cells underwent necrosis, contributing marginally to the overall cell death profile. In contrast, control HCT-116 cells exhibited minimal apoptosis, with a total apoptotic rate of 1.93%, comprising 0.59% early apoptosis and 0.14% late apoptosis. Necrosis was observed in only 1.2% of control cells.

The pronounced elevation in both early and late apoptosis following treatment suggests a potent pro-apoptotic effect of the T-1-PCPA loaded chitosomes. Given that apoptosis is a tightly regulated process responsible for the elimination of damaged or unwanted cells, the data indicate that the complex selectively induces programed cell death with minimal necrotic response.

The observed increase in early (21.55%) and total apoptosis (39.55%) suggests that T-1-PCPA-PC-CS induces programed cell death. Although mechanistic validation was not performed, it is plausible that the formulation activates the intrinsic mitochondrial apoptotic pathway, as reported with other methylxanthine analogs. This pathway typically involves mitochondrial outer membrane permeabilization, cytochrome c release, and downstream activation of caspase-9 and caspase-3.^89,90 Additionally, nanoparticle uptake may enhance intracellular drug accumulation, triggering oxidative stress or DNA damage that promotes apoptotic signaling.⁹¹ Future studies will focus on confirming these mechanisms using assays such as JC-1 staining for mitochondrial potential and caspase activity profiling.

Despite the encouraging in vitro findings, several limitations must be considered. A critical next step is in vivo validation to assess the pharmacokinetics, biodistribution, and therapeutic efficacy of the T-1-PCPA loaded chitosomes in a living system. The complexity of biological environments can significantly influence nanoparticle behavior, potentially affecting targeting efficiency and therapeutic outcomes. Moreover, while nanocarriers offer advantages in drug delivery, challenges such as immune clearance, opsonization, and accumulation in non-target tissues remain significant concerns in clinical translation. The long-term stability of the formulation under physiological conditions also requires thorough investigation to ensure consistent performance and shelf life. Finally, the potential for off-target cytotoxicity in normal tissues must be evaluated to confirm the selectivity and safety of the developed nanosystem.

Conclusion

This study evaluated the anticancer potential of a theobromine derivative, N-(4-Chlorophenyl)-2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-1-yl)acetamide (T-1-PCPA), encapsulated in chitosomes (T-1-PCPA-PC-CS). Molecular docking confirmed stable binding through the complex. The formulation was successfully prepared via thin film hydration and characterized by favorable colloidal properties, including a particle size of ∼142 nm, low PDI, high entrapment efficiency (89.2%), and a positive zeta potential. TEM confirmed spherical morphology. In vitro studies revealed significantly lower IC₅₀ values for the T-1-PCPA loaded chitosomes in HCT-116 and A549 cells compared to free drug and blank formulations, indicating enhanced cytotoxicity. The formulation also showed a higher selectivity index, suggesting reduced toxicity toward normal Vero cells. Cell cycle analysis demonstrated arrest at G0-G1, S, and G2/M phases. Flow cytometry showed increased total (39.55%) and early (21.55%) apoptosis. Cell migration assays confirmed reduced migration in HCT-116 cells. Overall, the T-1-PCPA loaded chitosomes demonstrated improved anticancer efficacy, selectivity, and functional impact on cancer cell behavior. These results support its further development as a promising targeted anticancer therapy.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X251384515 - Supplemental material for Exploring the Anticancer Potential of Chitosome-Encapsulated Theobromine Analogue: Integrated Computational, Physiochemical and In vitro Study

Supplemental material, sj-docx-1-npx-10.1177_1934578X251384515 for Exploring the Anticancer Potential of Chitosome-Encapsulated Theobromine Analogue: Integrated Computational, Physiochemical and In vitro Study by Ahmed M. Metwaly, Heba Abd-El-Azim, Mariam Zewail, Aisha A. Alsfouk, Eslam B. Elkaeed, Hazem Elkady and Ibrahim H. Eissa in Natural Product Communications

Footnotes

Acknowledgments

The authors would like to thank Research Center at AlMaarefa University for supporting this work.

ORCID iDs

Ahmed M. Metwaly

Hazem Elkady

Ethical Approval

Not applicable.

Statement of Informed Consent

Not applicable.

Funding

This research was funded by Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2025R116), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

All data pertaining to this research are included in the manuscript and .

Statement of Human and Animal Rights

Not applicable.

Sample Availability

Upon request, T-1-PCPA loaded chitosomes can be provided from the authors.

Supplemental Material

Contain detailed methodology, including tables, photos, and graphs, of the biological investigations, such as anticancer assays, selectivity studies, flow cytometry analysis, and migration assays.

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