Curcumin-Loaded Poly(xylitol sebacate) Nanoparticles Reduce Viability and Alter Morphology in Colorectal Cancer Cell Lines

1. Introduction

Colorectal cancer (CRC) is the third most common cancer diagnosed in the United States, with an estimated 158,850 new cases and 55,230 deaths projected in 2026 [1]. Polymeric nanoparticles are among the most versatile drug-delivery platforms because they improve solubility, protect payloads from premature degradation, and enable sustained or targeted release, improving efficacy while reducing systemic toxicity of poorly bioavailable therapeutics [2,3]. Biodegradable polyesters are particularly attractive carriers because they degrade into non-toxic byproducts while offering tunable degradation behavior and excellent biocompatibility [4,5]; although poly(lactic-co-glycolic acid) (PLGA) remains the most widely studied example, continued efforts target alternative polymer systems with greater synthetic versatility and architectural control [2,3,4,5,6].

Poly(xylitol sebacate) (PXS) is a biodegradable hyperbranched polyester synthesized by melt polycondensation of xylitol and sebacic acid. The five hydroxyl groups on xylitol drive extensive branching, producing a three-dimensional architecture with high terminal functional-group density that favors nanoparticle self-assembly, hydrophobic drug encapsulation, and future surface modification [4,5,7,8,9,10,11,12,13,14,15]. PXS also shows favorable biodegradation, biocompatibility, and a reduced inflammatory response relative to PLGA [10,11].

Our previous work established the synthesis, physicochemical characterization, and nanoparticle fabrication of hyperbranched PXS, identifying 15 h melt polymerization as optimal for reproducible nanoparticle formation. Curcumin-loaded PXS nanoparticles from that study had diameters of approximately 112 nm, strongly negative surface charge, 23.9% curcumin encapsulation efficiency, and sustained curcumin release under physiological conditions [10], establishing the materials foundation for biological evaluation.

Building on that platform, the present study evaluates whether PXS nanoparticles can effectively deliver curcumin to colorectal cancer cells and enhance its anticancer activity. Curcumin was selected as the model therapeutic for its well-documented antioxidant, anti-inflammatory, and anticancer activity, including suppression of proliferation and induction of apoptosis in CRC [2,3]; however, its clinical use is limited by poor water solubility, rapid degradation, and low bioavailability [4,5,6]. Polymeric nanoparticle encapsulation is a well-established strategy for overcoming these limitations [2,3,4,5,6]. Unloaded and curcumin-loaded PXS nanoparticles were therefore evaluated in four human CRC cell lines (HCT116, HT29, SW480, SW620) using cell-viability assays, phase-contrast microscopy, and fluorescence imaging. This work provides the first biological validation of PXS as a drug-delivery platform and represents the next step toward hyperbranched xylitol-based polyesters for anticancer applications.

2. Materials and Methods

2.1. Materials

Poly(xylitol sebacate) (PXS) was synthesized in-house by melt polycondensation of xylitol and sebacic acid under nitrogen at 150 °C, following Culpepper et al. [10]; the 15 h polymerization was used, as previously identified as optimal for nanoparticle fabrication. Curcumin (natural), dimethyl sulfoxide (DMSO), methanol, and Pluronic® F-127 were purchased from MilliporeSigma (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI-1640) medium, fetal bovine serum (FBS), penicillin streptomycin, phosphate buffered saline (PBS), paraformaldehyde, CellTiter-Blue® Cell Viability Assay reagent (Promega, Madison, WI, USA), and DAPI (Fisher Scientific, Waltham, MA, USA) were used as received. All aqueous solutions were prepared using deionized (DI) water.

2.2. Preparation of Curcumin-Loaded PXS Nanoparticles

Curcumin-loaded (LNP) and unloaded (UNP) PXS nanoparticles were prepared by nanoprecipitation, modified from Culpepper et al. [10] (Figure 1). Briefly, 0.03 - 0.05 g of PXS was dissolved in DMSO (LNP formulations additionally contained 1.2 mg curcumin) and added dropwise, under constant stirring, to 30 mL of an aqueous surfactant solution containing 1% w/v Pluronic® F-127 in a 1:2 (v/v) ratio of DI water to methanol. The suspension was probe sonicated using alternating one-minute on/off cycles for 20 minutes. One-mL aliquots were dispensed into 1.7-mL centrifuge tubes and centrifuged at 20,000 × g and 16 °C for 13 minutes; each pellet was washed twice more by resuspension in DI water followed by centrifugation (20,000 × g, 16 °C, 5 min). Washed pellets were pooled and resuspended in the final volume of 5 mL DI water. This reduced final suspension volume relative to our previously published procedure [10] increased nanoparticle and curcumin concentration per unit volume and reduced the risk of hypotonic effects during cell treatment (Section 3.3). Unloaded nanoparticles were prepared identically, omitting curcumin.

2.3. Nanoparticle Characterization

Nanoparticle morphology was evaluated by scanning electron microscopy (SEM; Phenom XL, High-Performance Materials Institute) after sputter-coating with 1 nm platinum (Cressington sputter coater). Hydrodynamic particle diameter was determined by dynamic light scattering (DLS; Lifesizer DLS 700) at 25 °C, and additionally after incubation at 37 °C for 24 and 48 h to confirm stability under the conditions used for the cell-viability assay. Curcumin loading was quantified using a Tecan NanoQuant Infinite M200 Pro plate reader; absorbance spectra (350 - 490 nm, average of 8 scans) were compared against a calibration curve generated from curcumin standards of known concentration (Figure S3) to calculate curcumin-equivalent nanoparticle doses. Zeta potential and encapsulation efficiency were not re-measured for the present batches; previously reported values [10] are used only as historical characterization of the PXS platform.

2.4. Cell Culture and Treatment Optimization

Human colorectal cancer cell lines HCT116, HT29, SW480, and SW620 were cultured at 37 °C in a humidified 5% CO₂ incubator in their recommended growth media supplemented with 10% FBS and 1% penicillin–streptomycin. Preliminary optimization in HCT116 cells (Figure S3) showed that nanoparticle treatment volumes equivalent to 30 μg/mL curcumin produced a hypotonic reduction in viability that was independent of nanoparticle toxicity, as confirmed using an equal-volume DI water control; 20 μg/mL was therefore selected as the working concentration, and the fabrication protocol (Section 2.2) was concentrated accordingly. Seeding density and exposure time were further optimized to 10,000 cells/well and 48 h. All four cell lines were subsequently treated under these optimized conditions.

2.5. Cell Viability, Morphology, and Fluorescence Imaging

Cell viability was evaluated using the CellTiter-Blue® assay. Cells (1 × 10⁴/well, black-wall 96-well plates) were treated with free curcumin, UNP, or LNP at an equivalent curcumin concentration of 20 μg/mL for 48 h, with untreated cells as controls; fluorescence was read on a Synergy H1 Multimode plate reader (Agilent Technologies) relative to untreated controls (n ≥ 3 independent replicates per condition). Phase-contrast images were collected after the 48 h treatment period to assess morphology. For fluorescence imaging, cells (2.5 × 10⁴/well, eight well chamber slides) were treated with free curcumin or LNP (20 μg/mL curcumin-equivalent, 24 h), fixed with 4% paraformaldehyde, and stained with DAPI (1:5000) prior to imaging on an Echo Revolve fluorescence microscope.

2.6. Statistical Analysis

Data are presented as mean ± standard deviation (n ≥ 3 independent biological replicates). Comparisons among treatment groups were performed by one-way ANOVA with Tukey’s multiple comparison test (GraphPad Prism v6, GraphPad Software, San Diego, CA, USA); p < 0.05 was considered significant, with exact p-values reported in figure legends.

3. Results and Discussion

3.1. Physicochemical Confirmation of the PXS Platform

Twenty-minute probe sonication produced homogeneous PXS nanoparticle suspensions, confirmed by DLS. Because this study extends the PXS platform previously established by Culpepper et al. [10], polymer identity was reconfirmed by ¹³C NMR, FTIR, TGA, and DSC (Figure S1). ¹³C NMR showed the expected sebacate methylene (~25 - 34 ppm), oxygenated xylitol (~61 - 73 ppm), and ester carbonyl (~173 - 175 ppm) resonances; FTIR showed residual hydroxyl (~3400 cm⁻¹), aliphatic –CH₂ (~2900 cm⁻¹), and ester C=O (~1700 - 1750 cm⁻¹) absorptions; TGA showed limited mass loss below ~200 °C followed by the principal degradation event; and DSC showed a subambient thermal transition without a prominent melting endotherm, consistent with an amorphous, hyperbranched structure. These results confirm that the PXS used here retains the chemical and thermal signature previously reported [10], allowing the present study to focus on nanoparticle behavior and biological performance.

3.2. Nanoparticle Size, Morphology, and Stability

SEM confirmed predominantly spherical morphology for both UNP and LNP, indicating that curcumin loading did not alter overall particle shape (Figure S2). DLS showed average hydrodynamic diameters of 212.40 nm (UNP) and 171.04 nm (LNP) at 24 h/25 °C (Figure S2); because DLS measures hydrated particles in suspension while SEM images dried particles, the two techniques were treated as complementary rather than directly equivalent. After incubation at 37 °C, the temperature and duration used for cell treatment, LNP measured 200.3 nm (24 h) and 162.10 nm (48 h), while UNP measured 202.50 nm (24 h) and 159.35 nm (48 h) (Table 1). Neither formulation showed the progressive size increase expected from aggregation, supporting nanoscale stability throughout the 48-h treatment window. Curcumin loading was quantified against a linear calibration curve (y = 0.0540x + 0.1258, R² = 0.9989; Figure S3) generated from 1–10 μg/mL curcumin standards, allowing curcumin-equivalent nanoparticle doses to be normalized across treatment groups.

3.3. Selection of a Non-Cytotoxic Working Concentration

Unloaded PXS nanoparticles were first tested at volumes equivalent to 10, 20, and 30 μg/mL curcumin-loaded nanoparticles in HCT116 cells. The 30 μg/mL volume produced a marked reduction in viability and crater-like morphology (Figure S4); an equal volume of DI water produced a comparable response, indicating a hypotonic effect of the treatment volume itself rather than nanoparticle cytotoxicity. Based on this, the nanoparticle fabrication protocol was concentrated (Section 2.2) and 20 μg/mL was adopted as the working concentration; at this dose, UNP produced no substantial reduction in viability or morphological change across all four cell lines (Figure S5), establishing that the PXS carrier itself is well tolerated and providing an appropriate baseline for evaluating curcumin-loaded nanoparticles.

3.4. Curcumin-Loaded Nanoparticles Increase Sensitivity of CRC Cells to Curcumin

At the 20 μg/mL curcumin equivalent dose, LNP produced a substantially greater reduction in viability than free curcumin across all four cell lines (Figure 2). Free curcumin alone produced only a modest (~20%) reduction in HCT116, SW480, and SW620, and essentially no reduction in HT29. In contrast, LNP reduced viability to ~30% (a ~70% reduction) in SW480 and SW620; a matched primary/metastatic cell-line pair from the same patient to ~60% (~40% reduction) in HT29 despite its limited response to free drug, and to ~70% (~30% reduction) in the most resistant line, HCT116. These viability changes were mirrored by phase-contrast morphology, where LNP treatment produced pronounced cell rounding and reduced cell density relative to free curcumin or UNP, while UNP produced no discernible morphological change in any line. Together, these results indicate that nanoparticle encapsulation markedly improves the cellular delivery and anticancer efficacy of curcumin in CRC cells, consistent with the physicochemical feasibility established previously for this platform [10], and that encapsulation can restore curcumin sensitivity in an otherwise poorly responsive line (HT29).

3.5. Fluorescence Imaging of Cellular Association

Fluorescence imaging in all four cell lines showed curcumin fluorescence (green) as sparse, punctuate signal under both free curcumin and LNP treatment (Figure 3). Free curcumin puncta were broadly distributed with limited apparent cell association, whereas some LNP associated puncta appeared closer to, or overlapping with, cell boundaries. However, signal intensity and puncta density were modest and qualitatively similar between conditions, and no co-localization analysis, flow cytometry, or dedicated internalization assay was performed. These images are therefore suggestive of, rather than definitive evidence for, enhanced cellular association of nanoparticle delivered curcumin, and are reported as a qualitative complement to the viability and morphology data.

4. Conclusions

This study provides the first biological validation of curcumin-loaded PXS nanoparticles as a delivery platform for colorectal cancer. Nanoparticles were reproducibly fabricated with the target size, spherical morphology, and 48-h colloidal stability required for cellular delivery and consistently outperformed free curcumin across four CRC cell lines. SW480 and SW620 — primary and metastatic lines from the same patient — were most sensitive; HT29 and HCT116 showed more modest but significant responses; and nanoparticle encapsulation restored sensitivity to curcumin in HT29, which was otherwise minimally responsive to the free compound. These findings are broadly consistent with reports of enhanced efficacy for other polymeric nanoparticle-delivered anticancer agents, including curcumin and other polyphenols [16,17] and 5-fluorouracil in colorectal and other cancer models [18,19]. Because this study did not include an apoptosis assay or cytoskeletal imaging, the mechanism underlying the observed morphological changes remains to be defined; future work incorporating apoptosis markers (e.g., Annexin V/caspase activity), cytoskeletal imaging, and in vivo efficacy studies is needed to further establish PXS as a biodegradable platform for anticancer drug delivery.