Enhanced Anticancer Activity of Ibuprofen and 2′-Hydroxy-2,3,5′-trimethoxychalcone-Linked Polymeric Micelles in HeLa Cells

Abstract

Cervical cancer (CVC) remains one of the most prevalent gynecological malignancies, necessitating the development of more effective therapeutic strategies to reduce tumor burden and prevent metastasis or recurrence. Conventional chemotherapy often causes severe side effects such as myelosuppression and leukopenia. This study aimed to develop a novel nanoparticle-based formulation to improve anticancer efficacy while minimizing toxicity. We synthesized a polymeric micelle formulation incorporating (S)-ibuprofen ((S)-IP) and 2′-hydroxy-2,3,5′-trimethoxychalcone (DK143), termed (S)-IP-DK143 NP. In vitro cytotoxicity assays were performed in HeLa cervical cancer cells. Mechanistic studies included intracellular reactive oxygen species (ROS) detection, analysis of mitochondrial dynamics, and assessment of mitogen-activated protein kinase (MAPK) pathway activation. Nanoparticle uptake was visualized using fluorescence microscopy. (S)-IP-DK143 NP demonstrated superior anticancer activity compared to the non-nanoparticle formulation, significantly reducing the IC₅₀ value in HeLa cells. The formulation induced ROS-mediated apoptosis, disrupted mitochondrial dynamics, and activated MAPK signaling, particularly via p38 phosphorylation. Fluorescence imaging confirmed rapid nanoparticle uptake within 1 h, suggesting efficient intracellular delivery. Our findings suggest that (S)-IP-DK143 NP enhances drug bioavailability and cytotoxicity through targeted delivery and ROS-mediated apoptotic signaling. The combination of pH-sensitive polymeric micelles and chalcone-based chemotherapy represents a promising approach for cervical cancer treatment. Further preclinical and clinical studies are warranted to validate its potential as an injectable anticancer formulation.

1. Introduction

Cervical cancer (CVC) is one of the most common gynecological malignancies and requires effective therapeutic strategies to reduce tumor burden before surgical intervention and to prevent metastasis or recurrence following surgery. However, current chemotherapy approaches often require high doses of anticancer agents, which are associated with severe adverse effects such as leukopenia and myelosuppression. Therefore, there is a critical need to develop therapeutic agents capable of providing potent anticancer activity at lower drug concentrations.

Previous studies have reported that chalcone-based compounds exhibit therapeutic potential against multiple cancer types, including nonsmall cell lung cancer, hepatocellular carcinoma, and ovarian cancer. Despite these promising findings, the high polarity of chalcone derivatives has been identified as a major limitation, resulting in low bioavailability and hindering their suitability for injectable formulations. Furthermore, most previously reported chalcone-based conjugates functioned as small molecules or prodrugs without an effective delivery platform for selective tumor accumulation. Recently, dual-drug systems and polymeric micelles have emerged as attractive strategies to improve solubility, pharmacokinetic behavior, and tumor selectivity through the enhanced permeability and retention (EPR) effect. To date, however, no study has evaluated an ibuprofen–chalcone conjugate incorporated into a polymeric micelle system capable of simultaneously exerting anti-inflammatory and pro-apoptotic effects with improved tumor targeting.

Advances in drug delivery technologiesincluding liposomes, nanoparticles, and polymeric formulationsdemonstrate that nanomedicine-based systems exhibiting the EPR effect can deliver therapeutic agents more efficiently than conventional small molecules within the tumor microenvironment. Nanoparticle formulations can improve drug accumulation and enhance therapeutic efficacy by exploiting tumor vasculature characteristics. For optimal micelle formation, the active pharmaceutical ingredient must possess amphiphilic properties that facilitate self-assembly. Previous research has shown that chalcone derivatives generally display poor aqueous solubility (typically <10–20 μg/mL in water) and low oral bioavailability, with plasma levels rarely exceeding 5–10% of the administered dose due to rapid metabolism and limited absorption. These physicochemical challenges significantly limit their clinical application, especially for parenteral delivery. Therefore, a micellar drug-delivery platform was selected in this study to improve solubility, stability, and bioavailability while enabling biocompatible cancer-targeted delivery.

In the present study, we synthesized polymeric micelles incorporating (S)-ibuprofen ((S)-IP) and 2’-hydroxy-2,3,5′-trimethoxychalcone (DK143), generating a dual-drug nanoparticle formulation termed (S)-IP-DK143 NP. We subsequently assessed its anticancer activity in cervical cancer cells and observed negligible cytotoxicity in normal cell lines, thereby supporting its overall biocompatibility. Our in vitro assays demonstrated that (S)-IP-DK143 NP significantly inhibited cervical cancer cell proliferation. Mechanistic analyses revealed that the compound induced cytotoxicity by promoting ROS-mediated mitochondrial dysfunction and modulating mitogen-activated protein kinase (MAPK) signaling, ultimately activating apoptotic pathways. Collectively, these findings suggest that (S)-IP-DK143 NP represents a promising therapeutic candidate for cervical cancer treatment.

2. Experimental Section

2.1. Materials

2-Hydroxy-5-methoxy acetophenone, 2,3-dimethoxy benzaldehyde, and (S)-1-phenylethylamine were purchased from TCl (Tokyo, Japan). Silica gel (70–230 mesh) was acquired from Sigma–Aldrich (St.Louis, MO, USA). Thionyl chloride, hydrochloric acid (HCl), magnesium sulfate (MgSO₄), methanol, dichloromethane (DCM), and pyridine were bought from Daejung (Seoul, Korea). mPEG–PLA (Molecular weight 4000 Da) was obtained from Samyang Biopharmaceuticals Corp. (Seoul, Korea). EX-Cytox was purchased from DOGEN (Seoul, Korea). Mito-tracker Red FM was acquired from Invitrogen (Waltham, MA, USA). Anti-P-p38, anti-T-ERK, anti-T-p38, and HRP-linked secondary antibodies [antimouse, antirabbit] were procured from Cell Signaling (Danvers, MA, USA). P-ERK and β-actin antibodies were supplied by Santa Cruz Biotechnology (Dallas, TX, USA). Miracle-Star Western Blot Detection System (ECL solution) was purchased from iNtRON Biotechnology (Gyeonggi-do, Korea). Dulbecco’s modified Eagle’s medium (DMEM) medium, Dulbecco’s phosphate buffered saline (DPBS), fetal bovine serum (FBS), and penicillin/streptomycin antibiotics used for cell culture were purchased from Welgene Inc. (Gyeongsangbuk-do, Korea). ¹H nuclear magnetic resonance (NMR) spectroscopy was performed using a 400-MHz instrument (Santa Clara, CA, USA). The Fourier transform infrared (FTIR, FT/IR-6300, Jasco Inc., Easton, MD, USA) spectra of the nanoparticles were obtained. The mean diameters and polydispersity of the micelles were measured using a Malvern Zetasizer Nano S90 (Malvern, Worcestershire, UK).

2.2. Preparation of (E)-2-(3-(2,3-dimethoxyphenyl)­acryloyl)-4-methoxyphenyl-2-(4-isobutyl phenyl)­propanoate ((S)-IP-DK143))-Loaded Polymeric Micelles

2.2.1. Synthesis of (E)-2-(3-(2,3-dimethoxyphenyl)­acryloyl)-4-methoxyphenyl-2-(4-isobutyl phenyl)­propanoate

(E)-3-(2,3-Dimethoxyphenyl)-1-(2-hydroxy-5-methoxyphenyl)­prop-2-en-1-one (DK143), one of the chalcone derivatives and (S)-ibuprofen, were synthesized using previously reported methods [15, 16]. DK143 and (S)-ibuprofen were used to synthesize (S)-IP-DK143 using the following procedures.

(S)-Ibuprofen (1.3 g, 6.30 mmol) and thionyl chloride (25 mL, 0.34 mol) were stirred at room temperature for 1 h. The mixture underwent fractional distillation to remove the thionyl chloride. DK143 (1.98g, 6.30 mmol), pyridine (1 mL, 0.012 mol), and dichloromethane (8 mL) were added to a mixture oil comprised of the acetyl chloride of ibuprofen. The mixture was stirred at room temperature for 3 h. At the end of the reaction, pyridine was removed from the mixture, which was then dissolved in dichloromethane with distilled water (8 mL) containing HCl (0.1 mL, 3.24 mmol). The liquid was purified by silica gel column chromatography to yield 51.2% (1.62g) of (S)-IP-DK143 as a yellow oil.

2.2.2. Preparation of (S)-IP-DK143-Loaded mPEG–PLA Nanoparticles (NPs)

(S)-IP-DK143 polymeric micelles were prepared using the thin-film hydration method. Briefly, 400 mg of mPEG–PLA and 100 mg of (S)-IP-DK143 were dissolved in 7 mL of dichloromethane, followed by rotary evaporation under reduced pressure at 40 °C. During this process, the resulting film contained (S)-IP-DK143 distributed in an amorphous form. The film was hydrated continuously in 17 mL of distilled water at room temperature. The solution was filtered through a 0.2 μm syringe filter and lyophilized for 48 h.

2.3. Characterization of the (S)-IP-DK143-Loaded mPEG–PLA Nanoparticles

2.3.1. Particle Size and Polydispersity (PDI) Analysis

The mean diameters and polydispersity of (S)-IP-DK143 polymeric micelles were measured using a Malvern-Zetasizer Nano S90 (Malvern, Worcestershire, UK). Dynamic light scattering (DLS) in the particle size analyzer was conducted at 633 nm and room temperature. The particle size was evaluated from the intensity distribution, and the particle size distribution is represented by the PDI (Table )

1. Characterization of (S)-IP-DK143 Nanoparticles.

	Drug loading amount
Drug carrier (Copolymer)	Drug:polymer (theoretical %)	Determined (%)	Encapsulation efficiency (%)	size (nm)	Zeta potential (mV)	Polydispersity index (PDI)
mPEG–PLA	1:6 (20%)	2.5%	97.8	82.34	1.93	0.082

2.3.2. Drug Encapsulation Efficiency (EE) and Drug Loading (DL)

UV/vis spectrophotometry (Optizen POP Bio, KLAB, Daejeon, Korea) determined the required concentrations of (S)-IP-DK143. The DL and EE of (S)-IP-DK143-loaded polymeric micelles were determined by diluting colloidal suspension samples in methanol. The diluted sample was prepared by dissolving 48 mg of the colloidal suspension samples in 10 mL of methanol (4800 ppm). A 1 mL solution sample was diluted in 9 mL of methanol (480 ppm). This process was repeated twice. The final concentration was 48 ppm. The absorbance of a 3 mL sample of a final diluted sample in methanol was measured at 215 nm. The DL and EE were calculated using the following formulas:

$$\mathrm{DL}\frac{\mathrm{Weight}\mathrm{of}\mathrm{drug}\mathrm{in}\mathrm{nanoparticle}}{\mathrm{Weight}\mathrm{of}\mathrm{feeding}\mathrm{polymer}\mathrm{and}\mathrm{drug}}\times 100\%$$

$$\mathrm{EE}\frac{\mathrm{Weight}\mathrm{of}\mathrm{drug}\mathrm{in}\mathrm{nanoparticle}}{\mathrm{Weight}\mathrm{of}\mathrm{drug}\mathrm{feeded}}\times 100\%$$

2.4. Biological Test

2.4.1. Cell Culture and Cytotoxicity Assay

HeLa cells were purchased from the Korea Cell Line Bank. The HeLa cells and normal cell line HEK293T were cultured in DMEM media containing 10% FBS and 1% penicillin–streptomycin at 37 ± 2 °C in a 5% CO₂ incubator. To determine the cell viability was determined by seeding HeLa cells (1 × 10⁴ cells/mL) into 96-well plates and treating them with diverse concentrations of IP-DK143 (0, 1, 3, 10, 30, and 100 μM), IP­(S)-DK143 (0, 1, 3, 10, 30, and 100 μM) and NP-IP­(S)-DK143 (0, 1, 3, 10, 30, and 100 μM), respectively. After 24 h, the HeLa cells were treated with 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2-H-5-tetrazolio]-1,3-benzene disulfonate-2 (WST-1) reagent and incubated at 37 ± 2 °C with 5% CO₂ for 2 h. The absorbance was measured using a microplate reader (wavelength at 450 nm).

2.4.2. Analysis of Mitochondria Morphology

HeLa cells (2 × 10⁵ cells/well) were seeded on a confocal dish and treated with 30 μM of IP­(S)-DK143 for 24 h. The HeLa cells were stained with Mito-Tracker. The mitochondria were observed using confocal microscopy (excitation 581 nm and emission 644 nm). The mitochondrial length was analyzed using ImageJ software.

2.4.3. Western Blotting

The mitogen-activated protein kinases (MAPKs) expression was analyzed by Western blotting using specific antibodies, such as antiphosphorylated extracellular signal-regulated kinase (P-ERK1/2), antitotal (T)-ERK1/2, anti-P-p38, anti-T-p38, and anti-β-actin. HeLa cells (1 × 10⁶ cells/well) were seeded at the 100 mm dish and treated with diverse concentrations of IP­(S)-DK143 (0, 1, 3, and 30 μM) or NP-IP­(S)-DK143 (0 and 3 μM) for 24h, respectively. The cell lysate was prepared by applying HeLa cells to the RIPA buffer. The protein lysates (30 μg) were boiled at 95 °C for 10 min. The proteins were separated by electrophoresis on 10% acrylamide gel. The separated proteins were transferred to a polyvinylidene fluoride membrane. The proteins were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature (RT) and incubated with each specific antibody in 3% BSA for 16 h at 4 °C. The membranes were incubated with a secondary antibody conjugated to horseradish peroxidase for 1 h at RT. The expression of the protein levels was analyzed using an ECL kit. The protein bands were quantified using the ImageJ software.

2.4.4. Cell Uptake Analysis

The mixture FTIC and NP-IP­(S)-DK143 was prepared to observe the cell uptake of NP-IP­(S)-DK143. The HeLa cells were stained with 4,6-diamidino-2-phenylindole (DAPI, excitation 359 nm and emission 461 nm) for 15 min at RT. The cell uptake was observed by confocal microscopy (LSM 780, Ziess, Germany).

2.4.5. Statistical Analysis

The data are expressed as the mean ± standard deviation of the mean of the indicated number of experiments. Statistical data analysis was performed using Student’s t-tests for comparing pairs of groups and ANOVA for multiple comparisons. P values <0.05 were considered significant

3. Results

3.1. Preparation of (S)-IP-DK143-Loaded Polymeric Micelles

(S)-IP-DK143 was synthesized from (S)-Ibuprofen and DK143, as shown in Scheme .

1. Synthetic Scheme Illustrating the Conjugation of (S)-ibuprofen with DK143.

The NMR spectra revealed a benzyl peak of ibuprofen and three methoxy peaks indicating (S)-IP-DK143 conjugation. To confirm the successful synthesis of the conjugated molecule (S)-IP-DK143, the individual monomers(S)-ibuprofen and DK143and the final conjugate were analyzed by ¹H NMR and FT-IR spectroscopy (Figure A–C). The ¹H NMR spectra of (S)-ibuprofen, DK143, and (S)-IP-DK143 (Figure B) showed distinct aromatic and aliphatic proton signals characteristic of each compound. Upon conjugation, the characteristic – CH₂OH proton resonance of DK143 at δ ≈ 3.7 ppm disappeared, while a new methylene signal corresponding to the ester linkage appeared at δ ≈ 4.1 ppm in (S)-IP-DK143. In addition, the aromatic proton peaks of (S)-ibuprofen shifted slightly downfield (δ ≈ 7.0–7.3 ppm), indicating deshielding due to ester bond formation. These changes confirm that the carboxyl group of (S)-ibuprofen reacted with the hydroxyl group of DK143 to form an ester bond. The FT-IR spectra (Figure C,D) further supported this conjugation. The broad – OH stretching band of DK143 near 3400 cm^–1 (Left circle) disappeared in the conjugate, while a strong new absorption band corresponding to the ester C = O stretch appeared at ∼ 1755 cm^–1 (Right circle, Figure E). Together, the ¹H NMR and FT-IR results clearly demonstrate the successful synthesis and structural integrity of (S)-IP-DK143.

1.

Structural characterization of synthesized compounds. (A) ¹H NMR spectrum of (S)-ibuprofen (IP). (B) ¹H NMR spectrum of DK143. (C) ¹H NMR spectrum and chemical structure of (S)-IP-DK143 showing disappearance of the DK143 – CH₂OH proton signal (∼3.7 ppm) and appearance of a new ester methylene signal (∼4.1 ppm), confirming successful esterification. (D) ¹³C NMR spectrum of (S)-IP-DK143 demonstrating characteristic carbon shifts consistent with the conjugated structure. (E) FT-IR spectrum indicating the loss of the hydroxyl stretch and the presence of a strong ester C = O absorption peak at 1755 cm^–1 (highlighted), further verifying conjugation of ibuprofen and DK143.

3.2. Drug Release Behavior

A drug release study was performed using the dialysis method to simulate both physiological (pH 7.2) and tumor microenvironmental (pH 5.7) conditions in phosphate buffer containing 7% Tween 80. The physicochemical characteristics of (S)-IP-DK143 nanoparticles, including particle size, polydispersity index, zeta potential, drug loading, and encapsulation efficiency, are summarized in Table . The surface morphology of the freeze-dried nanoparticles was examined by transmission electron microscopy (Figure A). The (S)-IP-DK143-loaded nanoparticles displayed a uniform spherical morphology with smooth and intact surfaces, showing no evidence of aggregation or collapse. As shown in Figure B, (S)-IP-DK143 exhibited a markedly pH-dependent release behavior, indicating that polymer degradation of the mPEG–PLA micelles was accelerated under acidic conditions. Within the first 24 h, approximately 19.5% of (S)-IP-DK143 was released at pH 5.7, whereas only ∼ 6% was released at pH 7.2 during the same period. The enhanced release at acidic pH can be attributed to the more rapid hydrolysis of the mPEG–PLA copolymer under low-pH conditions, consistent with the slightly acidic environment typically found in both primary and metastatic tumor tissues. These results confirm the successful formation and structural stability of the copolymeric micelles, as well as their suitability for sustained and pH-responsive drug release in tumor-mimicking conditions.

2.

Characterization and pH-responsive release behavior of (S)-IP-DK143 nanoparticles ((S)-IP-DK143 NPs). (A) Size distribution and Transmission electron microscopy (TEM, H-7100, Hitachi, Japan) image of (S)-IP-DK143-loaded NPs. (B) Drug release profile of (S)-IP-DK143 loaded-NPs. The release study was conducted at pH 7.2 and 5.7 with 7% tween 80 supplementation. The concentration of (S)-IP-DK143 released was quantified using UV/vis spectrophotometry.

3.3. Cell Cytotoxicity Effect of IP-DK143 and IP­(S)-DK143 on HeLa Cell

The cytotoxicity effect of the chalcone compounds, such as IP- DK143 and (S)-IP-DK143, was evaluated with a cell viability test using XTT assay. Normal cells and HeLa cells were treated with a range of concentrations (1, 3, 10, 30, and 100 μM) for 24 h. As shown in Figure A, both compounds exhibited minimal cytotoxicity in normal cells at concentrations below 30 μM, indicating acceptable biocompatibility within this range. And, the HeLa cells were treated with diverse concentrations of (S)-IP-DK143 (1, 3, 10, 30, and 100 μM) for 24 h. As shown in Figure B, treatment with racemic IP-DK143 induced a gradual reduction in cell viability with an IC_{5 0} value of 46.05 μM. In contrast, (S)-IP-DK143 exhibited a stronger cytotoxic effect with an IC_{5 0} value of 20.60 μM, indicating that the optical configuration significantly affects the biological activity of the compound (Figure C). Cells exposed to increasing concentrations of IP-DK143 or (S)-IP-DK143 displayed typical apoptotic morphological features, including cell shrinkage, rounding, and detachment from the culture surface. This study observed the cytotoxicity of both compounds. Considering the effects of the S form of IP-DK143, an isomer of IP, on the cytotoxicity, (S)-IP-DK143 was synthesized and tested in subsequent results. In this study, the (S)-IP-DK143 was more cytotoxic to HeLa cells than IP-DK143. Hence, a single isomer may increase the effectiveness of the target. Therefore, the new compound (S)-IP-DK143 is expected to improve bioavailability.

3.

Evaluation of cytotoxicity of IP-DK143 and (S)-IP-DK143 in normal and cancer cell lines. (A) Viability of normal cell line, HEK293T treated with IP-DK143 or (S)-IP-DK143 at concentrations of 0, 1, 3, 10, 30, and 100 μM for 24 h, showing minimal toxicity at lower concentrations. (B) Viability of HeLa cells treated with IP-DK143 at the indicated concentrations for 24 h. (C) Viability of HeLa cells treated with (S)-IP-DK143 at the same concentrations for 24 h. Cell viability was quantified using the WST-1 assay, and representative cell morphology images were obtained by microscopy. Data are presented as mean ± SD (n = X). *p < 0.05 vs untreated control.

3.4. IP-DK143 and (S)-IP-DK143 Induced the Mitochondria Dysfunction and Activated Apoptosis Related Proteins on HeLa Cells

In cancer cell death, excessive ROS production causes mitochondrial dysfunction. Ježek J et al. reported that ROS induces mitochondria fission in cancer cells. Mitochondrial morphology was observed using the Mito-tracker to confirm the changes in mitochondrial length. (S)-IP-DK143 reduced mitochondrial length significantly to 17.39 ± 4.6% compared to the untreated group, as shown in Figure A. Therefore, (S)-IP-DK143 exhibits anticancer efficacy by inducing intracellular ROS.

4.

Mitochondria morphological change and MAPK expression analysis by IP-DK143 and (S)-IP-DK143 on HeLa cells. (A) The cells were seeded (2 × 10⁵ cells/well) at the confocal dish and treated with diverse concentrations of IP-DK143 (30 μM) or (S)-IP-DK143 (30 μM) for 24 h. The cells were stained with Mito-Tracker dye and observed using confocal microscopy. The mitochondrial length was measured using ImageJ software. (B) HeLa cells were treated with various concentrations of (S)-IP-DK143 (0, 1, 3, and 30 μM) for 24h. The expression of MAPKs, such as the phosphorylation of ERK (P-ERK), total-ERK (T-ERK), P-p38, and p38, were analyzed using the specific antibodies. The quantification of P-ERK and P-p38 are expressed relative to the untreated (100%). All data are expressed as mean ± standard deviation. *p < 0.05 vs untreated group.

To further elucidate the downstream signaling associated with mitochondrial stress, Western blotting was performed to determine if (S)-IP-DK143 can regulate MAPK expression. As shown in Figure B, (S)-IP-DK143 (30 μM) significantly increased the level of p38 phosphorylation to 398.9 ± 24.8% compared with the untreated control, while total p38 levels remained constant. In contrast, the phosphorylation of ERK tended to decrease slightly, suggesting a shift in the MAPK balance toward p38-mediated apoptotic signaling. Therefore, (S)-IP-DK143 induced apoptosis through mitochondrial fission and p38 activation. This study tested whether chalcone compounds can induce P38 activity. Therefore, (S)-IP-DK143 induced apoptosis through mitochondrial fission and P38 activation.

3.5. (S)-IP-DK143 NP Increased the Cytotoxicity of HeLa Cells

To assess the biocompatibility of the designed formulation, (S)-IP-DK143 nanoparticles ((S)-IP-DK143 NPs) were first evaluated in normal cell lines at 24 and 48 h (Figure A). The results showed that concentrations above 100 μM induced noticeable cytotoxicity in normal cells, whereas lower concentrations demonstrated minimal toxicity. Based on these findings, subsequent experiments in HeLa cells were conducted using concentrations below this cytotoxic threshold.

5.

Cytotoxicity evaluation of (S)-IP-DK143 nanoparticles ((S)-IP-DK143 NPs) in normal and cancer cell lines. (A) Viability of normal cells treated with (S)-IP-DK143 NPs for 24 and 48 h, demonstrating minimal cytotoxicity at concentrations below 30 μM. HeLa cells were treated with diverse concentrations of (S)-IP-DK143 NP (0, 1, 3, 10, 30, and 100 μM) for 24h (B) and 48h (C). The cell viability was determined using the WST-1 reagent and microplate reader. The cell morphologies were observed under a microscope. All data are expressed as mean ± standard deviation. *p < 0.05 vs untreated group.

Next, to evaluate the anticancer efficacy of the formulation, IC_{5 0} values for (S)-IP-DK143 NPs were determined in HeLa cells using the XTT assay after 24 and 48 h of treatment. As shown in Figure B,C, (S)-IP-DK143 NPs exhibited a strong time-dependent cytotoxic response, with IC_{5 0} values of 2.553 μM at 24 h and 1.662 μM at 48 h. The progressive decrease in IC_{5 0} over time suggests sustained intracellular release of the active compound from the nanoparticle system, resulting in enhanced and prolonged cytotoxicity in cancer cells. Microscopic observations supported the quantitative findings, revealing dose-dependent morphological changes in HeLa cells. At lower concentrations, cells began to lose their polygonal shape and showed early apoptotic features, including cell shrinkage and partial detachment. At higher concentrations, extensive cell rounding, membrane disruption, and detachment were observed, indicating advanced stages of apoptosis and loss of viability. These morphological patterns were more pronounced after 48 h, consistent with the quantitative cytotoxicity data.

The hydrophobic nature of (S)-IP-DK143 limited its solubility in aqueous media, necessitating nanoparticle formulation to achieve stable dispersion and enhanced delivery efficiency. The incorporation of (S)-IP-DK143 into polymeric micelles effectively overcame its hydrophobicity, allowing improved solubility and controlled drug release. Therefore, (S)-IP-DK143 NPs not only enhanced the drug’s anticancer potency but also provided a formulation strategy suitable for intravenous administration, potentially improving its bioavailability and therapeutic efficacy in future applications.

3.6. (S)-IP-DK143 NP Induced Apoptosis on HeLa Cells

The intracellular uptake and molecular effects of (S)-IP-DK143 nanoparticles ((S)-IP-DK143 NPs) were examined in HeLa cells using fluorescence microscopy and Western blotting to elucidate their mechanism of action. As shown in Figure A, (S)-IP-DK143 NPs were efficiently internalized by HeLa cells within 1 h of exposure. Green fluorescence signals representing the FITC-labeled nanoparticles were clearly detected in the cytoplasmic region after 1 h and became more pronounced at 3 h, indicating progressive intracellular accumulation over time. This rapid uptake demonstrates the nanoparticles’ ability to effectively penetrate the cell membrane and deliver the encapsulated compound. It is consistent with previous reports showing that PLGA-based nanoparticles are rapidly endocytosed by cancer cells within 30–60 min, confirming the suitability of the micellar design for efficient cellular delivery.

6.

Analysis of (S)-IP-DK143 NP cell uptake and MAPK expression on HeLa cells. (A) The cells were seeded (2 × 10⁵ cells/well) at the confocal dish and treated with 3 μM of (S)-IP-DK143 NP. The nucleus was stained with DAPI, and cells were treated with FITC laded-NP-I (S)-IP-DK143. The cells were observed by confocal microscopy. The white arrows indicated the (S)-IP-DK143 NP. The graph provides quantitative analysis based on GFP expression levels in the cells. (B) The HeLa cells were treated 3 μM of (S)-IP-DK143 for 24 h. The expression of MAPKs, such as ERK (P-ERK), total-ERK (T-ERK), P-p38, and p38 phosphorylation, were analyzed using the specific antibodies. The quantification of p-ERK/T-ERK and p-p38/T-p38 are expressed relative to untreated (100%). All data are expressed as mean ± standard deviation. *p < 0.05 vs untreated group.

To further investigate whether (S)-IP-DK143 NPs modulate mitogen-activated protein kinase (MAPK) signaling, Western blot analysis was conducted to assess phosphorylation levels of ERK and p38 (Figure B). Treatment with (S)-IP-DK143 NPs markedly increased phosphorylation of p38, while the total p38 level remained unchanged, indicating selective activation of this pathway. In contrast, ERK phosphorylation was slightly reduced, suggesting a shift in MAPK signaling balance toward p38-mediated apoptosis. Quantitative densitometric analysis revealed that phosphorylated p38 levels increased to approximately 480% relative to control, confirming strong activation.

Collectively, these findings demonstrate that (S)-IP-DK143 NPs are rapidly internalized into HeLa cells and subsequently trigger apoptosis through p38 pathway activation. The correlation between nanoparticle uptake and MAPK signaling modulation suggests that efficient intracellular delivery is a key determinant of (S)-IP-DK143 NP–induced cytotoxicity. Therefore, the nanoparticle formulation not only enhances cellular uptake but also amplifies intracellular signal transduction leading to programmed cell death, validating (S)-IP-DK143 NP as a potent anticancer nanotherapeutic candidate.

4. Discussion

The development of ibuprofen and 2’-hydroxy-2,3,5′-trimethoxychalcone-linked polymeric micelles presents a promising approach for enhancing anticancer efficacy in HeLa cells. The drug release study revealed a pH-dependent behavior, with a significantly higher release rate at pH 5.7 compared to pH 7.2, suggesting that these micelles are well-suited for targeting the acidic tumor microenvironment (TME). This pH-sensitive release mechanism ensures that the drug is preferentially released in cancerous tissues, maximizing therapeutic efficacy while minimizing systemic toxicity.

In our study, cytotoxicity assays demonstrated that (S)-IP-DK143 exhibited stronger anticancer activity than its racemic counterpart, IP-DK143. The observed IC_{5 0} values indicate that the S-enantiomer significantly enhances cytotoxic effects in HeLa cells, consistent with prior reports on the superior biological activity of optically pure drug formulations. The rapid cellular uptake and improved cytotoxicity of (S)-IP-DK143 nanoparticles (NPs) further support their potential as a candidate for future preclinical and clinical development.

Reactive oxygen species (ROS) are well recognized for their ability to induce cancer cell death by damaging proteins, DNA, and other intracellular components. Apoptosis, however, is a multifactorial process that may involve additional signaling routes beyond ROS generation and mitochondrial fission. In this context, caspase activation, modulation of Bcl-2 family proteins, and endoplasmic reticulum stress signaling may also contribute to (S)-IP-DK143-induced cell death. Although our findings primarily demonstrate ROS-mediated mitochondrial dysfunction and p38 activation, these pathways may converge with both intrinsic and extrinsic apoptotic mechanisms. This broader mechanistic interpretation provides a more comprehensive understanding of the observed cytotoxic response and highlights the need for future studies to elucidate these complementary pathways in greater detail. Previous research has also emphasized the key role of mitochondrial dynamics in ROS-mediated apoptosis.

In addition, the potential synergy between ibuprofen’s anti-inflammatory activity and chalcone-induced ROS-mediated apoptosis further supports the therapeutic rationale of our system. Ibuprofen acts as a cyclooxygenase (COX) inhibitor, reducing prostaglandin production and thereby alleviating chronic inflammation that often contributes to tumor progression. On the other hand, chalcone derivatives promote oxidative stress and apoptosis through ROS generation and mitochondrial dysfunction. The integration of these two mechanisms may enhance the overall anticancer efficacy by simultaneously suppressing inflammation-driven tumor growth and promoting apoptotic signaling. This dual-action concept provides a strong scientific basis for the ibuprofen–chalcone hybrid micellar system as a multifunctional anticancer strategy.

Consistent with this mechanistic rationale, our data demonstrated that both IP-DK143 and (S)-IP-DK143 induce mitochondrial dysfunction and activate apoptosis-related proteins in HeLa cells. Specifically, (S)-IP-DK143 exhibited potent anticancer activity by increasing intracellular ROS, leading to mitochondrial damage and cell death. Further mechanistic evaluation revealed that (S)-IP-DK143 activates apoptotic pathways via p38 phosphorylation, and the observed mitochondrial fragmentation supports the involvement of mitochondrial dynamics in the apoptosis process. These findings align with current evidence highlighting the critical role of ROS-driven mitochondrial stress in cancer cell death.

The acidic TME, driven by altered metabolic phenomena such as the Warburg effect, represents an attractive therapeutic target. pH-responsive drug-delivery systemsincluding polymeric micelleshave been designed to exploit this feature by preferentially releasing therapeutic agents under acidic conditions. This strategy enhances drug accumulation at tumor sites while reducing systemic toxicity. For example, pH-low insertion peptides (pHLIPs) selectively deliver anticancer agents to tumors by inserting into cell membranes under acidic conditions, thereby improving therapeutic specificity and efficacy.

Our results demonstrate that (S)-IP-DK143 exerts potent anticancer activity by inducing intracellular ROS production, triggering mitochondrial dysfunction, and activating apoptosis-associated signaling pathways in HeLa cells. Given that the acidic TME further amplifies oxidative stress and mitochondrial instability, incorporating (S)-IP-DK143 into a pH-responsive delivery system may further enhance its therapeutic potential. The acidic milieu may accelerate drug activation, promote ROS accumulation, and amplify apoptotic signaling, thereby increasing specificity while reducing off-target toxicity.

Taken together, targeting the acidic TME using pH-sensitive polymeric micelles encapsulating (S)-IP-DK143 represents a highly promising approach for developing more effective and selective cancer therapies. This strategy aligns with the broader objective of exploiting tumor-specific vulnerabilities to improve therapeutic outcomes while minimizing harm to normal tissues. The development of (S)-IP-DK143 NPs offers a novel strategy to improve the bioavailability and anticancer potency of chalcone-based therapeutics. The combined features of pH-responsive drug release, potent cytotoxic activity, and targeted induction of apoptosis support its potential as an injectable treatment strategy for cervical cancer.

5. Conclusion

In this study, we synthesized a novel chalcone–ibuprofen hybrid compound, (S)-IP-DK143, and successfully formulated it into mPEG–PLA polymeric micelles to improve solubility, stability, and tumor-selective delivery. The resulting nanoparticles exhibited a pH-responsive release profile, with significantly accelerated drug liberation under acidic, tumor-mimicking conditions. In vitro assays demonstrated that (S)-IP-DK143 nanoparticles achieved markedly enhanced anticancer efficacy, reducing the IC_{5 0} by more than 10-fold compared with the free compound. Mechanistic analyses revealed that the nanomedicine induced apoptosis through ROS generation, mitochondrial dysfunction, and activation of p38 MAPK signaling. Importantly, the minimal cytotoxicity observed in normal cell lines supports its biocompatibility and safety potential for systemic administration. By integrating anti-inflammatory ibuprofen with pro-apoptotic chalcone chemistry in a single delivery platform, this dual-function nanoparticle system represents an innovative therapeutic strategy with strong translational potential for cervical cancer treatment. Collectively, these findings highlight (S)-IP-DK143 polymeric micelles as a promising nanomedicine candidate, warranting further pharmacokinetic profiling and in vivo efficacy studies to assess suitability for clinical development.

All data supporting the findings of this study are included within the article.

8.

S.B. and C.-W.Y. contributed equally to this work.

Conceptualization: S.J.B and C.-W.Y. Data curation: S.J.B. and C.-W.Y. Formal analysis: S.J.B., C.-W.Y., K.P.L., B.H.K., and E.W.J. Funding acquisition: S.H.K. Investigation: S.-H.L. and M.S.Y. Methodology: S.J.B., C.-W.Y., K.P.L., B.H.K., and E.W.J. Project administration: S.-H.L. and M.S.Y. Resources: S.J.B., C.-W.Y., K.P.L., B.H.K., and E.W.J. Supervision: S.-H.L., M.S.Y. Validation: S.J.B., C.-W.Y., K.P.L., B.H.K., and E.W.J. Visualization: S.J.B., C.-W.Y., K.P.L., B.H.K., and E.W.J. Writing (original draft): S.J.B. and C.-W.Y. Writing (review and editing): S.-H.L., M.S.Y., and Y.J.O.

No human participants or animals were involved in this study.

All authors read and approved the final manuscript

The authors declare no competing financial interest.