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. 2026 Feb 24;11(9):14847–14859. doi: 10.1021/acsomega.5c10664

TPGS and CS-Surface-Modified DPPC Liposomes Coloaded with Docetaxel and 5‑Fluorouracil: A Potential Avenue for Enhanced Colorectal Cancer Therapy

Abdullah H Alomrani †,‡, Mohamed M Badran †,‡,*, Turki Bin Duhaim †, Saad Alobid §, Wajhul Qamar §
PMCID: PMC12980442  PMID: 41835529

Abstract

Colorectal cancer (CRC) continues to be a foremost human health concern globally, with chemotherapy being the cornerstone of the therapy. However, conventional chemotherapy is often hampered by nonspecific targeting, limited efficiency, drug resistance, and undesirable adverse effects. 5-Fluorouracil (5-FU), the primary agent for CRC treatment, has a short duration of action and causes systemic toxicity. Docetaxel (DTX), another chemotherapeutic agent, has shown potential in CRC therapy, but its use is also hindered by poor targeting and resistance. Therefore, advanced drug delivery carriers can enhance their therapeutic efficacy. This combination offers a synergistic effect by targeting multiple pathways concurrently. This study aimed to design and assess dipalmitoylphosphatidylcholine (DPPC) liposomes (LPs) coloaded with DTX and 5-FU, surface-modified with d-α-tocopheryl poly­(ethylene glycol) succinate (TPGS-LPs) and chitosan (CS-LPs), as a dual-drug delivery for CRC therapy. Various LPs based on TPGS and CS, containing 5-FU and DTX, were successfully prepared and characterized by dynamic light scattering (DLS), and their entrapment efficiencies (EE%) were determined. The optimized formulations, DTX-TPGS-LPs, 5-FU-TPGS-LPs, and DTX/5-FU-TPGS-LPs, were subjected to in vitro release, cytotoxicity, and apoptosis studies using HT-116 cell lines. The LPs were successfully produced, with sizes ranging from 117.3 ± 2.7 to 205.6 ± 2.3 nm, and achieved EE% over 70% for DTX and 20% for 5-FU. The selected formulations, including DTX-TPGS-LPs, 5-FU-TPGS-LPs, and DTX/5-FU-TPGS-LPs, exhibited spherical morphology and controlled release profiles. Among these, the DTX/5-FU-TPGS-LPs formulation exhibited enhanced cytotoxicity and effectively induced apoptosis in HT-116 cells. These results highlight the potential of DTX/5-FU-TPGS-LPs as an effective dual-drug delivery system for CRC treatment; however, further optimization is required to balance its efficacy and toxicity for clinical application.

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1. Introduction

Cancer remains the leading cause of mortality worldwide, with colorectal cancer (CRC) being a major contributor to global cancer-related deaths. , Traditional chemotherapy, although widely employed, faces several challenges, including systemic toxicity, insufficient drug delivery to tumor sites, and the development of drug resistance. Despite its success in some cases, its efficacy is hindered by a lack of selectivity, which necessitates high doses and increases the risk of side effects. Therefore, there is an urgent need for an alternative strategy that improves therapeutic efficiency while minimizing side effects. One promising approach is combination therapy, in which multiple anticancer drugs are employed to target different pathways involved in cancer cell growth. This plan is more effective than single-drug therapies, as it reduces the risk of drug resistance and improves overall therapeutic outcomes. Such combined therapies have been successfully applied in clinical practice, particularly in the treatment of CRC.

CRC is a slowly progressing malignancy that originates as a polyp or abnormal growth in the lining of the colon or rectum. Among chemotherapeutic agents, 5-fluorouracil (5-FU) has demonstrated strong potential in CRC treatment. 5-FU is a pyrimidine analog that inhibits thymidylate synthase, disrupting DNA synthesis and inducing apoptosis. However, the clinical response to 5-FU is often suboptimal, with its efficacy hindered by low response rates. In addition, a significant obstacle in cancer therapy is the development of multidrug resistance (MDR). Therefore, high doses are necessary, which increase the risk of side effects such as gastrointestinal and cardiotoxicity. The combination of 5-FU with other anticancer agents has excellent potential to overcome these limitations and MDR, thereby improving its effectiveness. Combining 5-FU with docetaxel (DTX) could enhance the antitumor efficacy and prevent the development of MDR. , DTX is a taxane chemotherapeutic agent that inhibits microtubules, leading to mitotic arrest and apoptosis. DTX has demonstrated efficacy against various human malignancies, including breast, cervical, small and non-small cell lung, prostate, colorectal, and stomach cancers. Therefore, DTX is a promising candidate for further therapeutic combinations. DTX has significant limitations, including poor water solubility, nonspecific distribution, and dose-limiting toxicity. The combination of DTX and 5-FU, given their distinct mechanisms of action, holds great promise for improving treatment efficacy in CRC. Furthermore, an effective drug delivery system is required to overcome these problems and augment the performance of payload-loaded drugs. Liposomes (LPs) have the potential to increase the efficacy of various marketed drugs. However, to maximize their therapeutic potential, continuous optimization is required to achieve greater stability and drug efficacy. For effective delivery, 5-FU and DTX were formulated into LPs modified with d-α-tocopheryl poly­(ethylene glycol) 1000 succinate (TPGS) and chitosan (CS). Many studies have reported that the surface-modified LPs improve the therapeutic efficacy of anticancer drugs. It has been confirmed that TPGS can overcome MDR by inhibiting P-glycoprotein (P-gp) activity, a membrane efflux transporter responsible for drug resistance. For example, TPGS-coated LPs exhibited a significant enhancement in the cytotoxicity of DTX against MDR lung cancer cells (A549/DDP) compared with conventional and pegylated LPs. The cellular uptake test showed the highest dye intensity in the cytoplasm with coumarin-6-loaded TPGS-coated liposomes compared with traditional and pegylated liposomes. Similarly, Han et al. reported that TPGS-LPs have a positive impact on paclitaxel activity. The cellular uptake and cytotoxicity in MDR breast cancer cells. The cellular uptake of paclitaxel-loaded TPGS-coated LPs was increased by 3 to 5-fold compared to paclitaxel-loaded LPs against MCF-7/ADR cells. In addition, Song et al. also found that TPGS-LPs enhanced the bioavailability and therapeutic efficacy of Ziyuglycoside I in the treatment of myelosuppression. Likewise, Kumari et al. showed that doxorubicin-loaded TPGS-LPs could serve as effective targeted nanocarriers for tumor treatment.

Moreover, CS-LPs have also received attention for their positive charge and mucoadhesive properties, which promote cellular uptake of anticancer drugs. Gil-Gonzalo et al. highlighted the efficacy of CS-LPs in improving the bioavailability of ciprofloxacin and etoposide. These results highlight the potential of TPGS-LPs and CS-LPs to enhance the efficiency of loaded drugs. Similarly, Alomrani et al. confirmed the effectiveness of 5-FU-loaded CS-LPs against the human colon cancer cell line. Moreover, Alshraim et al. demonstrated that DTX-loaded CS-coated LPs exhibited superior anticancer efficacy against HT29 CRC. Moya-Garcia et al. further showed that etoposide-loaded CS-LPs significantly increased cytotoxicity in U373 glioblastoma cells. These studies demonstrate the potential of TPGS-LPs and CS-LPs as effective drug carriers.

These findings provide a strong rationale for the continued exploration of surface-modified LPs to optimize anticancer treatment strategies. This study presents a triple-strategy approach: codelivery of 5-FU and DTX, a robust LPs platform for drug loading, and surface modification with TPGS and CS, which has not been reported previously. The surface modification of LPs represents a significant advancement in drug delivery, enhancing cellular uptake and tumor localization. This multifunctional platform produces a targeted, efficient, and synergistic approach to maximize therapeutic efficacy against CRC cells.

2. Materials and Methods

2.1. Materials

Docetaxel (DTX) was purchased from Hangzhou Hyper Chemical Co., Ltd. (Zhejiang, China). 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC) was supplied by Lipoid GmbH (Ludwigshafen, Germany). 5-Fluorouracil (5-FU), low molecular weight chitosan (50–190 kDa, viscosity20–300 cP), cholesterol (Chol), d-α-tocopheryl poly­(ethylene glycol) 1000 succinate (TPGS), and MTT (3-(4,5-dimethyl-thiazol-2-yl)- 2,5-diphenyltetrazolium bromide) were purchased from Sigma-Aldrich Chemical Co., Ltd. (St. Louis, MO). Tween 80 and glacial acetic acid were obtained from BDH Organic (Poole, Dorset, U.K.). Methanol and acetonitrile (HPLC grade) were provided by Fisher Scientific Co. (Loughborough, U.K.). HCT-116 cells were sourced from the American Type Culture Collection (ATCC, Manassas, VA). All other chemicals used were of analytical grade.

2.2. Preparation of DTX and 5-FU-Loaded TPGs-LPs and CS-LPs

The lipid film hydration method, with minor modification, was used to prepare different types of LPs. , DPPC and Chol were dissolved in chloroform/methanol (2:1) in a round-bottom flask. The organic solvent mixture was evaporated under reduced pressure at 65 °C using a rotary evaporator to form a thin lipid film, which was then further dried by purging with nitrogen for 5 min. For TPGS-LPs, TPGS was added to the lipid mixture during film formation. The resulting dry film was then hydrated with 10 mL PBS (pH 7.4; 65 °C) under vortexing for 30 min. , CS-coated liposomes (CS-LPs) were obtained by adding a 0.2% (w/v) CS solution (in 0.1% acetic acid) to the liposome dispersion at a 1:1 volume ratio. The resulting mixture was stirred for 30 min at room temperature to facilitate electrostatic surface modification. , For coloaded surface-modified LPs, DTX was added to the organic phase, and 5-FU to the aqueous phase, simultaneously. Subsequently, the dispersions were probe-sonicated for 3 min (60% power) in an ice bath to reduce vesicle size, followed by centrifugation at 5000 rpm for 5 min to remove undissolved and large particles. The obtained LPs were then stored at 4 °C for further use.

The final preparations were designated as DTX-LPs, DTX-TPGS-LPs, and DTX-CS-LPs for DTX; 5-FU-LPs, 5-FU-TPGS-LPs, and 5-FU-CS-LPs for 5-FU; DTX/5-FU-LPs, DTX/5-FU-TPGS-LPs, and DTX/5-FU-CS-LPs for combined formulations (Table ).

1. Composition of DTX, 5-FU, and Their Co-Loaded Conventional LPs, TPGs-LPs, and CS-LPs .

type of LPs DPPC (molar) chol (molar) TPGS (molar) CS (mg/mL) DTX (mg/mL) 5-FU (mg/mL)
DTX-LPs 7 3 0 0 1 0
DTX-TPGS-LPs 7 3 1 0 1 0
DTX-CS-LPs 7 3 0 1 1 0
5-FU-LPs 7 3 0 0 0 1
5-FU-TPGS-LPs 7 3 1 0 0 1
5-FU-CS-LPs 7 3 0 1 0 1
DTX/5-FU-LPs 7 3 0 0 1 1
DTX/5-FU-TPGS-LPs 7 3 1 0 1 1
DTX/5-FU-CS-LPs 7 3 0 1 1 1
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DPPC, dipalmitoylphosphatidylcholine; Chol, cholesterol; TPGS, d-α-tocopheryl poly­(ethylene glycol) succinate; CS, chitosan; DTX, docetaxel; FU, 5-Fluorouracil; LPs, liposomes.

2.3. Vesicle Size Distribution and ζ-Potential Measurements

All formulations were analyzed using a Malvern Zetasizer (Nano ZS; Malvern Instruments Ltd., Worcestershire, U.K.) to determine average vesicle size, polydispersity index (PDI), and ζ-potential. For each measurement, 0.1 mL of the sample was diluted 100-fold with distilled water. Measurements were performed in triplicate, with five runs per sample at room temperature. Data are expressed as the mean of three replicates.

2.4. Entrapment Efficiency and Drug Loading Measurements

An indirect ultracentrifugation method was used to determine the entrapment efficiency (EE%) of DTX and 5-FU. , To separate the unentrapped drugs, 1 mL of each formulation was centrifuged at 40,000 rpm for 30 min at 4 °C using an Optima Max-E Ultra-Cooling Centrifuge (Beckman Coulter, Pasadena, CA). In addition, the actual drug content was determined by dissolving 1 mL of each sample in methanol and sonicated in a water bath for 30 min to disrupt the vesicles. The concentrations of DTX and 5-F in supernatants (free drug) and disrupted samples (total drug) were quantified by HPLC. Each experiment was conducted three times. The following equation was used to calculate EE% and DL%

EE(%)=total drug−free drugtotal drug×100 1
DL(%)=amount of the drug in formulationstotal amount of formulations×100 2

2.5. HPLC Analysis

In a separate analysis, DTX quantification was measured using reverse-phase HPLC. A 20 μL of DTX sample was injected and analyzed. The mobile phase consisted of acetonitrile and water (55:45, v/v), and the mixture was eluted through a reverse-phase C18 column (μ Bondapak, 4.6 mm × 150 mm, 10 μm particle size) at 1 mL/min at room temperature. Peak detection was performed using a UV detector set to 227 nm.

The quantification of 5-FU was performed using reverse-phase HPLC, as previously described, with minor modifications. The mobile phase consisted of 40 mM phosphate buffer, which was adjusted to pH 7.0 with 10% w/v KOH. A volume of 20 μL from each sample was injected, with the mobile phase delivered through a reversed-phase C18 column (Bondapak, 4.6 mm × 150 mm, 10 μm particle size) at a flow rate of 1 mL/min. The concentration of 5-FU was detected using a UV detector set at 260 nm. The HPLC system was monitored using Empower software (Waters). The details of the HPLC analytical method, along with representative chromatograms and calibration curves, are provided in the Supporting Information as Table S1 and Figures S1–S4.

2.6. In Vitro Release Study

The in vitro profiles of DTX-TPGS-LPs, 5-FU-TPGS-LPs, and DTX/5-FU-TPG-LPs, selected for their high EE, were assessed using the dialysis bag method. One milliliter of each formulation was transferred to a preswelled dialysis membrane (12–14 kDa MwT cutoff) and immersed in 50 mL of phosphate-buffered saline (PBS; pH of 7.4), containing 0.5% w/v Tween 80 to maintain sink conditions. The beakers were incubated in a shaking water bath at 37 °C and 100 rpm for 48 h. At predetermined time points, aliquots from the medium were withdrawn and replaced with an equal volume of fresh PBS. HPLC was used to determine the drug concentrations of the collected samples, and the cumulative release of DTX and 5-FU was expressed as the percentage of drug released over time. All experiments were carried out in triplicate. The release was fitted to zero-order, first-order, Higuchi, and Korsmeyer-Peppas models to identify the predominant release kinetics.

2.7. Morphological Characterization

Transmission electron microscopy (TEM) was used to visualize the morphology of DTX-TPGS-LPs, 5-FU-TPGS-LPs, and DTX/5-FU-TPGS-LPs, which were chosen for their high EE%. A drop of each LPs was placed on a copper grid, and excess liquid was gently blotted with filter paper. The samples were then stained with a 2% uranyl acetate solution and air-dried in a dust-free environment. After drying, the vesicles were imaged using TEM at 200 kV.

2.8. Short-Term StabilityTop of Form

The short-term stability of DTX-TPGS-LPs, 5-FU-TPGS-LPs, and DTX/5-FU-TPGS-LPs at 4 °C after 1-, 2-, 3-, and 4-week storage was evaluated to assess their potential for practical applications. , The stored samples were examined for changes in the particle size.

2.9. In Vitro Cytotoxicity Study

An in vitro cell viability assay was conducted to evaluate the anticancer potential of TPGS-LPs against colon cancer cells (HCT-116). The study compared the effects of free drugs (DTX and 5-FU), blank TPGS-LPs, DTX-TPGS-LPs, 5-FU-TPGS-LPs, and combined DTX/5-FU-TPGS-LPs. HCT-116 cells were cultured in DMEM supplemented with 10% FBS and 1% pen-strep. Cells were seeded at 104 cells/well in 96-well plates in 200 μL of medium and incubated at 37 °C in a 5% CO2 atmosphere for 24 h to allow cell attachment. After that, the cells were exposed individually to different concentrations of DTX (0.02–1 μM) and 5-FU (0.2 μM-20 μM) as single agents for 24 and 48 h. For the combination therapy, a molar ratio of DTX to 5-FU (1:10) was used. Untreated cells maintained under the same conditions served as the negative control. Following treatment, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well for 4 h. The medium was then removed, and 200 μL of DMSO was added to dissolve the formazan. Absorbance was measured at 550 nm using a microplate reader (Bio-Tek Instruments Inc., Winooski, VT). Each experiment was conducted three times. The cell viability was calculated using the following equation

cell viability(%)=treated celluntreated cell×100 3

2.10. The Combination Index for DTX and 5-FU

To evaluate the combination index of the free DTX and 5-FU, HCT-116 cells were seeded in 96-well plates (104 cells/well) and incubated at 37 °C in a 5% CO2 for 24 h. The cells were exposed to increasing concentrations of DTX (0.01–2.0 μM), 5-FU (0.1–20.0 μM), and their combination at a 1:10 ratio for 48 h. After treatment, the medium was removed, and 100 μL of MTT solution was added to each well. The plate was then incubated for 2 h. Formazan crystals were solubilized in DMSO, and absorbance at 550 nm was measured using a microplate reader to assess cellular viability. The drug interaction was analyzed with CompuSyn software using the Chou-Talalay method. The CI < 1 indicates synergy, CI = 1 denotes an additive effect, and CI > 1 indicates antagonism.

2.11. In Vitro Apoptosis Assay

The apoptotic activity of DTX and 5-FU-loaded TPGS-LPs on HCT-116 cells was evaluated by flow cytometry using Annexin V fluorescein isothiocyanate (Annexin V-FITC)/ Propidium Iodide (PI) staining. Annexin V-FITC binds specifically to phosphatidylserine, an early indicator of apoptosis, as it translocates from the inner to the outer plasma membrane. Additionally, PI, a fluorescent DNA-binding dye, distinguishes between viable and dead cells, staining the cell in late apoptosis and necrosis. HTC-116 cells were seeded in 6-well plates (1 × 105 cells/well) in 2 mL of medium and incubated for 24 h. The cells were treated with free DTX, 5-FU, blank TPGS-LPs, DTX-TPGS-LPs, 5-FU-TPGS-LPs, and combined DTX/5-FU-TPGS-LPs (DTX at 0.025 μM and 5-FU at 0.25 μM) and incubated at 37 °C. Untreated cells were utilized as the control. After 24 h, the cells were gently collected, washed with PBS, and resuspended in 100 μL of Annexin V-FITC. They were incubated at 4 °C for 15 min, and 5 μL of PI was added in the dark for an additional 15 min. Subsequently, Annexin V binding buffer (400 μL) was added, and the sample was analyzed by flow cytometry (Beckman Coulter).

2.12. Statistical Analysis

All data obtained from the study were analyzed using the Microsoft Excel software 2010. One-way ANOVA test to compare groups for a single parameter, and p < 0.05 was considered statistically significant.

3. Results and Discussion

Multidrug resistance (MDR) remains a significant obstacle to the effectiveness of chemotherapy, reducing the efficacy of many anticancer agents. Combination chemotherapeutics is a recognized strategy to enhance treatment outcomes. 5-FU is a well-established anticancer drug characterized by multiple mechanisms, including inhibition of apoptosis, alteration of the cell cycle, and repair of DNA damage. DTX has been reported to resensitize tumors to 5-FU and produce synergistic effects against colon cancer due to different mechanisms of action. To maximize the therapeutic efficacy of this combination, surface-modified LPs were designed to codeliver DTX and 5-FU. TPGS has been successfully used to functionalize LPs, improving the therapeutic results of loaded drugs. Moreover, CS modification further enhances cellular uptake and drug efficacy. Therefore, TPGS- or CS-modified LPs serve as effective carriers for the targeted delivery of DTX and 5-FU.

3.1. Vesicle Size and ζ-Potential Measurements

The vesicle size distribution and ζ-potential of LPs are important measurable parameters that influence their stability, in vivo and in vitro behaviors. LPs with diameters less than 200 nm have been shown to enhance drug transport to cancer cells. The vesicle size, PDI, and ζ-potential values of the prepared liposomes are presented in Table and Figure . The average vesicle size of DTX- and 5-FU-loaded LPs varied with formulation. The average particle size values of DTX-loaded liposomes were 120.2 ± 2.5 nm for DTX-LPs, 145.4 ± 3.4 nm for DTX-TPGS-LPs, and 154.3 ± 4.6 nm for DTX-CS-LPs. Similarly, the vesicle size values of 5-FU-loaded formulation were 117.3 ± 2.7 nm for 5-FU-LPs, 164.6 ± 7.1 nm for 5-FU-TPGS-LPs, and 170.3 ± 4.4 nm for 5-FU-CS-LPs. The dual-loaded DTX and 5-FU showed an increase in their particle size, measuring 145.5 ± 5.3 nm for DTX/5-FU-LPs, 198.8 ± 3.6 nm for DTX/5-FU-TPGS-LPs, and 202.5 ± 2.3 nm for DTX/5-FU-CS-LPs. These results revealed that the particle size values of the surface-modified LPs were larger than those of the unmodified LPs. The magnitude of the size increase of CS-LPs was higher (P < 0.001) than that reported with TPGS (TPGS-LPs) and LPs (P < 0.001). The observed increase in particle size could be attributed to the formation of a thick, hydrated CS layer on the LP surface. , In addition, the strong electrostatic attraction to negatively charged LPs may significantly enhance the hydrodynamic diameter. The increase in vesicular size of TPGS-LPs can be attributed to the integration of TPGS into the lipid bilayer, which expands the bilayer thickness. In parallel, dual loading of DTX and 5-FU into TPGS-LPs resulted in a significant increase in vesicle size compared with the corresponding single-drug loading (P < 0.001). This enlargement is expected when hydrophobic (DTX) and hydrophilic (5-FU) drugs are coentrapped in TPGS-LPs, thereby altering lipid packing density.

2. Particle Size, PDI, and ζ-Potential Measurements of DTX and 5-FU-Loaded Surface-Modified Liposomes .

formulations particle size (nm) PDI ζ-potential (mV)
DTX-LPs 120.2 ± 2.5 0.088 ± 0.062 –2.06 ± 0.62
DTX-TPGS-LPs 145.4 ± 3.4 0.253 ± 0.081 –5.29 ± 0.39
DTX-CS-LPs 154.3 ± 4.6 0.302 ± 0.036 8.20 ± 0.28
5-FU-LPs 117.3 ± 2.7 0.135 ± 0.040 –2.45 ± 0.44
5-FU-TPGS-LPs 164.6 ± 7.1 0.384 ± 0.026 –7.01 ± 0.93
5-FU-CS-LPs 170.2 ± 4.4 0.343 ± 0.044 10.43 ± 1.10
DTX/5-FU-LPs 145.5 ± 5.3 0.287 ± 0.064 –1.52 ± 0.47
DTX/5-FU-TPGS-LPs 198.8 ± 3.6 0.385 ± 0.045 –6.34 ± 0.56
DTX/5-FU-CS-LPs 205.6 ± 2.3 0.391 ± 0.061 7.97 ± 0.71
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Each value represents the mean ± SD (n = 3)

1.

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Particle size distribution of (A) DTX-LPs, (B) DTX-TPGS-LPs, (C) DTX-CS-LPs, (D) 5-FU-LPs, (E) 5-FU-TPGS-LPs, (F) 5-FU-CS-LPs, (G) DTX/5-FU-LPs, (H) DTX/5-FU-TPGS-LPs, and (I) DTX/5-FU-CS-LPs.

As a consequence of this structural change, a bimodal size distribution was observed for certain formulations, including 5-FU-TPGS-LPs and DTX/5-FU-TPGS-LPs. This effect is likely formulation-related and may be attributed to incomplete size reduction during sonication, which produces heterogeneous vesicle populations. Insufficient sonication energy may partially break larger LPs into smaller ones, leading to coexisting populations. Furthermore, entrapment of a hydrophilic drug such as 5-FU within the aqueous core of LPs is inherently challenging because it tends to partition into the internal water phase. This behavior may further contribute to nonuniform vesicle formation and size heterogeneity.

PDI is a commonly used parameter for evaluating the particle-size distribution of liposomal vesicles. According to Malvern Zetasizer guidelines, a PDI value <10% indicates a monodisperse distribution, whereas 10–20% indicates a moderate size distribution. However, a PDI > 30% reflects a polydisperse population with size heterogeneity. The PDI of the unmodified liposomes ranged from 0.088 to 0.287 (Table ), indicating a homogeneous size distribution within an acceptable range. While the PDI values of the surface-modified liposomes are slightly higher (0.253–0.391), indicating moderate heterogeneity. This effect may be attributed to the additional coating layer, which can influence vesicle formation and size.

The surface charge of the obtained LPs plays a key role in determining their therapeutic performance. This charge is influenced by lipid composition, surface-coating materials, and the drug entrapped within LPs. The prepared LPs exhibit a neutral or slightly negative ζ-potential due to the neutral nature of phospholipids. However, incorporating TPGS and CS can affect ζ-potential values (Table ). TPGS may form a compact, hydrophilic layer on LPs, thereby increasing LP negativity, solubilization, and drug-delivery efficacy. , Despite TPGS lacking intrinsic ionic properties, it imparts a modestly negative ζ-potential of TPGS-LPs. This is because TPGS is an amphiphilic surfactant; it changes surface characteristics, particularly the LPs charge. The negative charge of TPGS-LPs can be attributed to the PEG moiety and succinate groups in TPGS, which form a hydrophilic shell on the liposomal surface. In aqueous media, the carboxylate (−COO−) group in succinate can ionize, resulting in a negative charge. Consequently, the ζ-potential of TPGS-modified LPs becomes more negative compared to unmodified LPs. In addition, CS-LPs exhibited a positive value of ζ-potential, which can be attributed to positively charged CS. CS, as a cationic polysaccharide, induces a marked shift in the liposomal surface charge from negative to positive. This change is due to electrostatic interactions between the negatively charged phospholipid bilayer and the positively charged amino groups of CS. CS, as a cationic polysaccharide, induces a marked shift in the liposomal surface charge from negative to positive. This change is due to electrostatic interactions between the negatively charged phospholipid bilayer and the positively charged amino groups of CS. This positive charge has been observed in many studies, in which CS-LPs exhibited positive ζ-potentials. ,,

3.2. Entrapment Efficiency and Drug Loading Measurements

High EE% and DL% can result in effective therapeutic outcomes when using chemotherapeutic agents. In this study, both DTX and 5-FU were evaluated across different LPs formulations (Figure ).

2.

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EE% of DTX (A), 5-FU (B), the DL% of DTX (C), and 5-FU (D) in LPs, TPGS-LPs, CS-LPs, and their combined formulations. Each value represents the mean ± SD (n = 3).

For DTX, TPGS-LPs achieved the highest EE% (93.8%) and DL% (8.5%), followed by CS-LPs at 82.9% and 7.4%, respectively. On the other hand, the conventional LPs exhibited the lowest EE% of 74.1% and DL% of 6.7% (Figure A,C). A similar pattern was observed for 5-FU: TPGS-LPs showed the highest EE% of 34.2% and DL% of 2.9%, followed by CS-LPs at 26.9% and 2.2%, and conventional LPs at 20.4% and 1.8% (Figure B,D). These results clearly highlight the effect of surface modification on the drug EE%, with TPGS significantly increasing EE% and DL% compared to conventional LPs and CS-modified LPs. Interestingly, the coloading of DTX and 5-FU led to a noticeable reduction in EE% and DL% for each drug. In conventional DTX/5-FU-LPs, both EE% and DL% for DTX decreased to 67.6% and 5.6%, respectively. Similarly, for 5-FU-LPs, the EE% decreased to 18.9% and the DL% to 1.4% (p < 0.05). However, DTX/5-FU-TPGS-LPs maintained relatively high EE% and DL% values, with 91.7% and 7.1% for DTX, and 31.4% and 2.7% for 5-FU, respectively. While EE% and DL% values of DTX/5-FU-CS-LPs were 77.4% and 5.9% for DTX and 23.1% and 2.0% for 5-FU, respectively (Figure C,D).

These results confirmed the significant role that surface modification, particularly TPGS, plays in optimizing the EE% and DL% of DTX and 5-FU. Variations in EE% and DL% values may be attributed to several factors, including differences in lipid packing, drug-lipid interactions, and surface charge modification (TPGS and CS). The amphiphilic nature of TPGS-LPs is particularly notable, as it enhances drug solubility, thereby increasing their loadings. , In contrast, coloading of DTX and 5-FU reduced EE% and DL% for each drug due to the competition between the two drugs for limited sites within the lipid bilayer or aqueous core of the LPs. Additionally, the simultaneous incorporation of both hydrophobic and hydrophilic drugs could disrupt optimal lipid packing and bilayer structure, further contributing to the EE%.

3.3. In Vitro Release Study

The release profiles of chemotherapeutic drugs significantly influence their intracellular uptake and therapeutic efficacy. Surface-modified LPs can alter the release kinetics of the drugs, making them a promising choice for sustained release. This study examines the release pattern of 5-FU and DTX from TPGS-LPs, which were selected for their high EE (Figure ). In the case of DTX formulations, the DTX suspension exhibited a minimal release profile, with only 8.8 ± 2.6% released after 6 h and 27.9 ± 3.1% after 48 h (Figure A). This slow release is attributed mainly to the lipophilic nature of DTX, which limits its diffusion in aqueous medium. In contrast, DTX-TPGS-LPs demonstrated a marked improvement in the release of DTX, with 19.7 ± 2.9% released after 6 h and 63.5 ± 4.2% after 48 h. When DTX coloaded with 5-FU in TPGS-LPs, the release of DTX was slightly slower compared to DTX-TPGS-LPs, with 11.1 ± 3.7% released after 6 h and 53.1 ± 3.9% after 48 h. This indicates that the presence of 5-FU may affect its diffusion within LPs structure. The release of 5-FU was evaluated from its solution, TPGS-LPs, and coloaded TPGS-LPs, as shown in Figure . The 5-FU solution exhibited rapid release, reaching 100% within 6 h. This is mainly due to the hydrophilic nature of 5-FU, which facilitates rapid release. However, the release of 5-FU from TPGS-LPs was delayed, with 64.4 ± 5.6% after 6 h and 80.9 ± 5.6% released after 48 h. The TPGS acts as a barrier, slowing the release of 5-FU across the lipid bilayer. DTX/5-FU-TPGS-LPs exhibited a slower release profile compared to 5-FU-TPGS-LPs, with 52.9 ± 7.4 after 6 h and 71.8 ± 7.4% released after 48 h. The hydrophobic nature of DTX may interfere with the diffusion of 5-FU within the LPs matrix. Moreover, the reduction in EE% of DTX/5-FU-TPGS-LPs might further explain the observed decrease in drug release.

3.

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Drug release profiles of DTX suspension, DTX-TPGS-LPs, 5-FU solution, 5-FU-TPGS-LPs, and coloaded DTX/5-FU-TPGS-LPs. Each value represents the mean ± SD (n = 3).

In addition, the release kinetics of DTX and 5-FU, each alone and in combination loaded into TPGS-LPs, were evaluated using zero order, first order, Higuchi, and Korsmeyer-Peppas models. The correlation coefficient values (R 2) and the release exponent (n) values were calculated to predict the release behavior (Table ). For DTX-TPGS-LPs (both single or combined), the Korsmeyer-Peppas release model was the best fit, with the highest R 2 values. The n values ranged from 0.580 to 0.652, indicating a non-Fickian transport mechanism, governed by both diffusion and relaxation. For the 5-FU solution, the R 2 values followed first-order kinetics (R 2 = 0.9210), suggesting a concentration-dependent release pattern. , The release of 5-FU from TPGS-LPs (a single and combined) followed the Korsmeyer-Peppas release model, with n values ranging from 0.149 to 0.221. These values suggest a Fickian diffusion-controlled release. Several factors, including the properties of entrapped drugs, particulate sizes, and LP compositions, can contribute to the kinetic model. TPGS-LPs are characterized by prolonged drug release, making them particularly beneficial for drug delivery.

3. In Vitro Release Kinetics Model of Single DTX Susps., 5-FU-Solution, DTX/5-FU-Loaded TPGS-LPs.

formulations zero order first order Higuchi Korsmeyer-Peppas n value
DTX Formulations
DTX-Sups. 0.8471 0.8943 0.9519 0.9765 0.624
DTX-TPGS-LPs 0.7736 0.9418 0.9509 0.9638 0.580
DTX/5-FU-TPGS-LPs 0.8491 0.9468 0.9199 0.9528 0.652
5-FU Formulations
5-FU-Solution 0.6299 0.9210 0.8033 0.8236 0.174
5-FU-TPGS-LPs 0.6316 0.9067 0.8166 0.9814 0.149
DTX/5-FU-TPGS-LPs 0.7002 0.9304 0.8779 0.9523 0.221
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3.4. Stability Study

DTX-TPGS-LPs, 5-FU-TPGS-LPs, and DTX/5-FU-TPGS-LPs that demonstrated the highest EE% were selected to evaluate their physical stability. TPGS-LPs were kept at 4 °C, and their stability was assessed by monitoring changes in particle size at 0, 2, 3, and 4 weeks (Figure ). The findings revealed a minor and gradual increase in particle size. This increase suggests some degree of particle agglomeration or fusion over time. However, the particles remained within an acceptable size range, indicating that TPGS-LPs remained stable.

4.

4

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Short-term physical stability of DTX-TPGS-LPs, 5-FU-TPGS-LPs, and DTX/5-FU-TPGS-LPs at 4 °C.

3.5. TEM Morphology

The TEM of DTX-TPGS-LPs, 5-FU-TPGS-LPs, and DTX/5-FU-TPGS-LPs revealed moderately uniform particles with spherical shape (Figure ). The noticeable difference in particle size between TEM images and dynamic light scattering (DLS) results was attributed to vesicle shrinkage during drying, which led to smaller vesicle sizes.

5.

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TEM images of (A) DTX-TPGS-LPs, (B) 5-FU-TPGS-LPs, and (C) DTX/5-FU-TPGS-LPs (scale bar: 200 nm).

3.6. In Vitro Cytotoxicity Study

The cytotoxic activity was performed on HCT-116 cells using different concentrations of free DTX, free 5-FU, DTX-TPGS-LPs, 5-FU-TPGS-LPs, and DTX/5-FU-TPGS-LPs (Figure ). The study demonstrated that the plain TPGS-LPs exhibited minimal cytotoxicity, with cell viability >90% in HCT-116 cells after 24 and 48 h, indicating their safety (Figure S5). Cell viability decreased for all tested samples with increasing concentrations. As depicted in Figure A, the treatment with 1 μM of free DTX resulted in 67% and 55% cell viability at 24 and 48 h, respectively. In comparison, 20 μM of free 5-FU resulted in 72% and 63% cell viability at 24 and 48 h, respectively (Figure B). When these drugs were loaded into TPGS-LPs, a notable enhancement in cytotoxic activity was observed relative to free drug solutions (Figure A,B). DTX-TPGS LPs exhibited a more potent cytotoxic effect compared to 5-FU-TPGS LPs. The HCT-116 cell viability after treatment with 1 μM of DTX-TPGS-LPs was 42% and 24% at 24 and 48 h, respectively. Treatment with 20 μM 5-FU in TPGS-LPs reduced cell viability to 45% and 32% at 24 and 48 h, respectively (Figure A,B). The DTX/5-FU-TPGS-LPs exhibited a greater cytotoxic effect than their solution-form combination. The cell viability of HCT-116 cells treated with free DTX and 5-FU at a 1:10 molar ratio was 49% and 44%, respectively, at 2 μM DTX and 20 μM 5-FU after 24 and 48 h. However, further reduction in cell viability was observed when HCT-116 cells were treated with DTX/5-FU coloaded TPGS-LPs at 2 μM DTX and 20 μM 5-FU, reporting 25% and 11% at 24 and 48 h, respectively (Figure C). These results revealed that coloading DTX and 5-FU into TPGS-LPs augments the cytotoxic effects against HCT-116 cells. Such enhancement in cytotoxicity could be attributed to the liposomes’ role in improving the cellular uptake of loaded drugs.

6.

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Cell viability of HCT-116 cells treated with DTX, and DTX-TPGS LPs (A), 5-FU and 5-FU-TPGS LPs (B), and DTX/5-FU and DTX/5-FU-TPGS-LPs (C) over 24 and 48 h. Each value represents the mean ± SD (n = 3).

The IC50 of free DTX after 24 and 48 h of incubation was 1.58 and 0.82 μM, respectively. When DTX was prepared in TPGS-LPs, these values decreased to 0.17 and 0.04 μM after 24 and 48 h, respectively. Similarly, the IC50 values at 24 and 48 h of free 5-FU were 42.64 and 24.45 μM, and they decreased to 7.73 and 5.71 μM after 5-FU was incorporated into TPGS-LPs. In the case of combination therapy, free DTX/5-FU and DTX/DTX/5-FU-TPGS-LPs displayed IC50 values of 0.75 μM and 0.014 μM for DTX after 48 h, respectively, and 10.04 μM and 0.13 μM for 5-FU. These results demonstrate the positive impact of TPGS-modified LPs on the anticancer activity of 5-FU and DTX in combined therapy. TPGS may enhance the cytotoxic activity of the loaded drug by inhibiting P-gp, thereby reducing drug efflux and increasing intracellular drug levels. , TPGS is an amphiphilic polymer that increases membrane permeability and drug endocytosis. TPGS can also disrupt mitochondrial metabolism, increasing ROS production, which triggers the apoptotic pathway of cell death. In addition, TPGS increases the solubility of a hydrophobic drug. The high cytotoxicity of coloaded TPGS-LPs is promising for CRC treatment. The significant cytotoxic effect of the coloaded TPGS-LPs formulation was also confirmed in supplemented images (Figure S5) compared with 5-TPGS-LPs and DTX-TPGS-LPs.

The clinical feasibility of combining DTX and 5-FU has been well established through Phase I and II trials across multiple cancer types, including gastric, breast, and head and neck cancers. − Furthermore, a pharmacokinetic study reported no significant interactions between these drugs, indicating that they can be coadministered without altering each other’s pharmacokinetic profiles. These results support the continued use of the DTX-5-FU combination, which holds promise for enhancing antitumor efficacy.

3.7. Synergistic Cytotoxic Effect of Free DTX and 5-FU

HCT-116 cells treated with free DTX and 5-FU showed the inhibition of cancer cell proliferation in a dose-dependent manner with IC50 values of 0.82 μM and 24.45 μM, respectively, after 48 h of treatment. When the cells were treated with combinations of DTX and 5-FU at a 1:10 molar ratio for 48 h, the IC50 values decreased to 0.75 and 10.04 μg/mL, indicating a synergistic cytotoxicity. In addition, the dose–effect curve for DTX and 5-FU treatment, analyzed using the median-effect plot, showed a CI < 1 for the combination, indicating synergistic effects of DTX and 5-FU (Figure A). The dose–response index calculated using CompuSyn software also showed DRI values >1 for the DTX-5-FU combinations (Table and Figure B), suggesting synergistic effects of these drugs. In addition, the cytotoxicity of each drug at high dose, when administered individually, was higher than that of the drug combination. Therefore, the combination of DTX and 5-FU is more effective at lower doses in killing cancer cells. Therefore, DTX potentiated the effect of 5-FU and vice versa. The synergistic cytotoxicity of combined 5-FU and DTX may be attributed to the potentiated toxic effect in cancer cells, arising from their diverse mechanisms of action, which could enhance the tolerability profile of these drugs. Furthermore, the dual-loaded nanocarriers can facilitate drug delivery and augment their efficacy. Furthermore, the impact of surface-modified LPs on the cellular permeation of DTX and 5-FU was also studied.

7.

7

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Graphical representations obtained from the CompuSyn report for free combined DTX/5-FU with their incubation with HCT-116 cells after 48 h. The Combination Index (CI) (A), Dose Response Index (DRI) (B). CI < 1 and DRI > 1, indicating a synergistic effect of the drugs.

4. Data Showing the CI and DRI Values for Free DTX and 5-FU against HCT-116 Cells .

5-FU (μM) DTX (μM) CI value DRI DTX DRI DTX
0.1 0.01 0.39941 3.23909 11.0278
0.25 0.025 0.36483 3.24874 17.5372
2.5 0.25 0.91289 1.20078 12.4850
5 0.5 0.71022 1.49376 24.5294
10 1 0.54715 1.89739 49.7266
20 2 0.51175 2.00529 76.4882
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a

CI: Combination Index; DRI: Dose Response Index.

3.8. Flow Cytometry Assay

The findings revealed that the apoptotic effect predominated across all treated groups, whereas the necrotic effect was minimal (Figure ). Remarkably, the combination of DTX and 5-FU resulted in a higher percentage of early apoptotic cells and fewer late apoptotic cells than the individual treatments. It was observed that free 5-FU induced early apoptosis in 1.3% of cells, whereas 5-FU-TPGS-LPs induced apoptosis in 2.8%. However, the effects on late apoptosis were insignificant in cells treated with free 5-FU and 5-FU-TPGS-LPs (1.2% and 0.2%, respectively). A notable apoptotic effect was observed with cells treated with DTX formulations. DTX-TPGS-LPs improve early apoptosis by 13.5% relative to free DTX (7.3%) without a change in late apoptosis rates. Most potent apoptotic activity was observed with the coloaded TPGS-modified LPs (DTX/5-FU-TPGS-LPs), in which 14.2% total apoptosis was obtained, which was higher than that obtained by the combined free drugs (10.1%). These findings suggest that the potential effect can be achieved when DTX and 5-FU are administered together. This behavior is consistent with previous studies conducted on DTX in combination with another chemotherapeutic. The DTX/-FU-TPGS-LPs exhibited a high reduction of HCT-116 cell growth and apoptotic activity due to the difference in their mode of action. DTX induces mitotic arrest, while 5-FU disrupts DNA synthesis, increasing caspase activation and p53-mediated apoptosis. Their combination may amplify DNA damage and enhance mitochondrial apoptosis. Furthermore, the TPGS enhances cellular uptake, thereby maximizing therapeutic activity.

8.

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Effect of free DTX, DTX-TPGS-LPs, free 5-FU, 5-FU-TPGS-LPs, DTX/5-FU-TPGS-LPs, and plain TPGS-LPs formulations on apoptosis. HCT-116 cells were stained with FITC Annexin V/Propidium Iodide after 24 h of treatment.

This study presents several limitations that should be addressed in future research. First, the long-term stability of the formulations at 37 °C (the typical physiological temperature) was not assessed. Such data are important for understanding how the formulation would behave in vivo over a long time. However, an initial stability study was conducted at 4 °C for one month as a preliminary step to identify early signs of the instability before progressing to longer-term studies. Another limitation concerns drug-release testing across different pH media to understand how the drug is released under various physiological conditions. The drug-release study was performed only in phosphate-buffered saline (PBS) at pH 7.4, which reflects physiological conditions for drug release.

4. Conclusion

In this study, TPGS-modified LPs coloaded with DTX and 5-FU were successfully fabricated and appraised for their anticancer efficacy against the HCT-116 colorectal cancer cell line. The optimized formulations, DTX-TPGS-LPs, 5-FU-TPGS-LPs, and DTX/5-FU-TPGS-LPs, exhibited nanoscale sizes and negative ζ-potentials. They were spherical in shape and demonstrated a controlled release profile for both drugs over 48 h, with 49.02 ± 3.64% and 35.3 ± 8.0% of DTX and 5-FU, respectively. Notably, the DTX/5-FU-TPGS-LPs showed potent anticancer and apoptotic effects in HCT-116 compared with the single-drug formulation. These findings confirmed the synergistic efficacy of the combination of DTX and 5-FU. These results highlight the potential of TPGS-modified LPs for codelivering DTX and 5-FU, thereby improving therapeutic efficacy in colorectal cancer treatment.

Future studies should focus on evaluating the long-term stability of DTX- and 5-FU-loaded TPGS-LPs at 37 °C, as well as conducting in vivo studies to assess pharmacokinetics and therapeutic efficacy. Additionally, further studies are needed to elucidate the interaction mechanisms of these formulations to optimize their clinical potential

Supplementary Material

ao5c10664_si_001.pdf (715.4KB, pdf)

Acknowledgments

The authors extend their appreciation to the Ongoing Research Funding Program (ORF-2025-1008), King Saud University, Riyadh, Saudi Arabia.

All the data are available throughout the manuscript.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c10664.

  • HPLC chromatograms of DTX and 5-FU; calibration curve of DTX; calibration curve of 5-FU; cell viability of HCT-116 cells treated with plain TPGS-LPs over 24 and 48 h; morphological evaluation of free DTX, DTX-TPGS-LPs, free 5-FU, 5-FU-TPGS-LPs, DTX/5-FU-TPGS-LPs, and plain TPGS-LPs formulations on HCT-116 cells; FT-IR spectra of 5-FU, DTX, TPGS-LPs, 5-FU-TPGS-LPs, DTX-TPGS-LPs, and DTX/5-FU-TPGS-LPs (PDF)

Conceptualization, A.H.O.; methodology, M.B.; software, M.B.; validation, T.D.; formal analysis, S.A.O.; investigation, M.B.; resources, T.D. and M.B.; data curation, A.O.; writing-original draft preparation, M.B.; writingreview and editing, W.Q. and S.O.; visualization, M.B.; supervision.

The authors declare no competing financial interest.

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Supplementary Materials

ao5c10664_si_001.pdf (715.4KB, pdf)

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All the data are available throughout the manuscript.

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