Synthesis, characterisation and evaluation of polyethylene glycol–β-cyclodextrin–curcumin–zinc oxide nanoparticles for mosquitocidal, antibacterial and anticancer applications

Abstract

To synthesise and characterise polyethylene glycol–β-cyclodextrin–curcumin–zinc oxide nanoparticles (PEG–Beta-Cur–ZnO NPs) and evaluate their stability, anti-mosquitocidal, antibacterial, and anticancer activities for potential biomedical and vector-control applications. Characterisation was performed using UV–Vis spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), and Transmission Electron Microscopy (TEM). Stability was assessed over time and pH ranges. Biological activity was evaluated through anti-malarial larval toxicity assays, adult mosquito longevity and fecundity studies, antibacterial assays against Escherichia coli and Staphylococcus aureus, and anticancer tests on HeLa cells measuring ROS generation, cell viability, DNA damage, and mitochondrial membrane potential. UV–Vis spectra confirmed successful functionalization with peak shifts to 423 nm. Stability studies showed time-limited dispersion (up to ~ 90 min) but improved stability under acidic conditions. FTIR revealed characteristic O–H, C=O, and Zn–O bands, confirming conjugation. XRD analysis indicated a preserved hexagonal wurtzite structure with high crystallinity. TEM imaging showed quasi-spherical morphology (76 nm). In larval assays, PEG–Beta–Cur–ZnO NPs achieved near-complete mortality at lower doses compared to free curcumin, while adult mosquitoes exhibited reduced lifespan and fecundity. Histopathology confirmed severe midgut damage in treated larvae. Antibacterial testing demonstrated superior inhibition zones and elevated ROS generation with PEG–Beta–Cur–ZnO NPs, indicating enhanced bacterial killing. In HeLa cells, the formulation induced significant ROS production, increased cell death, DNA fragmentation, and mitochondrial dysfunction compared to curcumin and ZnO NPs alone. PEG–Beta–Cur–ZnO NPs exhibit effective surface functionalization, pH-sensitive stability, and significantly enhanced larvicidal, antibacterial, and anticancer activities. These multifunctional nanoparticles show strong potential for eco-friendly vector control and biomedical applications.

Introduction

Globally, infectious diseases and cancer remain significant public health challenges. In 2022, malaria accounted for approximately 249 million infections and 608,000 fatalities globally, with the majority of cases concentrated in sub-Saharan Africa¹. Bacterial infections, especially those involving multidrug-resistant strains, continue to threaten healthcare systems, causing millions of deaths annually². Cancer remains one of the foremost causes of death globally, accounting for an estimated 9.7 million deaths in 2022, with its incidence expected to rise significantly in the coming years³. There is, therefore, an urgent need for multifunctional, effective, and safe therapeutic agents capable of addressing these diverse health challenges.

Nanotechnology, the manipulation of matter at the nanometer scale (1–100 nm), offers transformative solutions across medicine, energy, and environmental science. Nanoparticles (NPs), owing to their large surface area relative to volume, adjustable chemical and physical characteristics, and ability to be functionally modified, have shown great potential as drug delivery platforms^4–6. Among metal oxide nanoparticles, zinc oxide (ZnO) NPs are particularly attractive because of their biocompatibility, intrinsic antimicrobial activity, ease of synthesis, and potential for photodynamic and anticancer applications^7,8. However, the bare surface of ZnO NPs can lead to aggregation and potential toxicity, necessitating surface modification strategies to improve their biomedical applicability⁹.

Curcumin, a naturally occurring polyphenol extracted from the rhizome of Curcuma longa (turmeric), exhibits a wide range of bioactivities, including antioxidant, anti-inflammatory, antimicrobial, and anticancer properties attributable to its unique chemical structure. This structure comprises two aromatic rings linked by a seven-carbon chain featuring an α, β-unsaturated β-diketone moiety, which underlies its functional^10,11. However, despite its pharmacological potential, curcumin’s clinical application is significantly limited by poor water solubility, chemical instability, rapid metabolic degradation, and low systemic bioavailability¹². Nanoparticle-based delivery systems, particularly surface-functionalized nanoparticles, have been extensively explored to overcome these limitations, improving curcumin’s solubility, stability, targeted delivery, and controlled release^13,14.

To overcome these limitations, nanoparticle-based delivery systems have been explored to improve curcumin’s solubility, stability, targeted delivery, and controlled release. In particular, β-cyclodextrin, a cyclic oligosaccharide with a hydrophobic cavity and hydrophilic exterior, has been widely used to form inclusion complexes with poorly soluble drugs. This encapsulation improves aqueous solubility, chemical stability, and bioavailability by shielding the drug from environmental degradation and enhancing its dispersibility¹⁵. Polyethylene glycol (PEG) is widely used as a biocompatible and hydrophilic polymer for nanoparticle surface modification. PEGylation reduces nonspecific protein adsorption, enhances colloidal stability, prolongs systemic circulation time, and minimizes immunogenicity^16,17. Functionalizing ZnO nanoparticles with PEG and β-cyclodextrin inclusion complexes synergistically enhances the anticancer properties of the nanomaterials¹⁸.

This study aims to develop and characterize polyethylene glycol–β-cyclodextrin–curcumin–zinc oxide nanoparticles (PEG–Beta–Cur–ZnO NPs), investigate their physicochemical properties and stability, and evaluate their enhanced anti-malarial, antibacterial, and anticancer activities.

Materials and methods

Chemicals

Zinc acetate dihydrate, sodium hydroxide, PEG, beta-cyclodextrin, and curcumin were procured from Sigma-Aldrich (USA). All other chemicals and solvents were of analytical grade.

Preparation of ZnO NPs

ZnO NPs were synthesized via a precipitation method¹⁹. Briefly, 0.1 M zinc acetate solution was prepared in deionized water and stirred vigorously at 60 °C. A 0.2 M sodium hydroxide solution was added dropwise until pH 10 was achieved. The resulting white precipitate was aged for 2 h, washed repeatedly with deionized water and ethanol, and dried at 80 °C overnight. The dried powder was calcined at 400 °C for 2 h to obtain crystalline ZnO NPs.

Preparation of β-cyclodextrin–curcumin inclusion complex

The Beta–Cur was prepared by the method of Arya and Raghav²⁰, with modification. Curcumin (0.1 g) was dissolved in 10 mL of ethanol under constant stirring. Separately, β-cyclodextrin (1 g) was dissolved in 40 mL of deionized water at room temperature. The ethanolic curcumin solution was added dropwise to the aqueous β-cyclodextrin solution with continuous stirring for 2 h. The mixture was then sonicated for 30 min to facilitate complex formation. The resulting solution was stirred overnight at room temperature. The inclusion complex was collected by freeze-drying and stored in a desiccator until use.

Synthesis of PEGylated ZnO NPs

ZnO NPs (0.5 g) were dispersed in 50 mL of deionized water containing 1% (w/v) PEG. The suspension was sonicated for 30 min to ensure uniform dispersion and PEG coating. The mixture was then stirred at room temperature for 4 h to achieve PEGylation. The PEGylated ZnO NPs were collected by centrifugation at 8000 rpm for 10 min, washed twice with deionized water to remove unbound PEG, and dried at 60 °C.

Functionalization with β-cyclodextrin–curcumin inclusion complex

The PEGylated ZnO NPs (0.5 g) were resuspended in 50 mL of deionized water containing the previously prepared β-cyclodextrin–curcumin inclusion complex (0.1 g). The mixture was sonicated for 30 min to aid in the adsorption of the inclusion complex onto the nanoparticle surface. The solution was then stirred for 4 h at room temperature to ensure effective binding. The functionalized PEG–β-CD–Cur–ZnO NPs were collected by centrifugation at 8000 rpm for 10 min, washed with deionized water to remove excess unbound complex, and dried under vacuum.

The drug loading efficacy was calculated by two ways, first based on indirect method by estimating the curcumin content of the supernatant and second based on direct estimation of the curcumin content present in the pellet obtained after centrifugation. The drug concentration in supernatant and redispersed pellets was determined by measurements of its UV absorbance at 470 nm using UV/visible spectroscopy and the percentage loading of curcumin onto nanoparticles were estimated by the following formula.

Where, wo is the weight of curcumin conjugated on the PEG–Beta–ZnO, w is the weight of PEG–Beta–ZnO nanoparticles.

Characterization

UV−Visible spectra were recorded using a Shimadzu UV-2600 spectrophotometer in the 200–800 nm range. Stability over time (0–180 min) was assessed by monitoring changes in absorbance. pH-dependent stability was evaluated by dispersing nanoparticles in buffer solutions (pH 2.5, 4.5, 6.5, 8.5, and 10.5) and recording spectral shifts (4). FTIR spectra were recorded using a Bruker Tensor 27 spectrometer in the range 4000–400 cm⁻¹ using KBr pellets. XRD patterns were obtained on a Rigaku Ultima IV diffractometer using Cu Kα radiation (λ = 1.5406 Å) operating at 40 kV and 30 mA. Diffraction angles (2θ) from 20° to 80° were scanned at 2°/min. The average crystallite size of the ZnO nanoparticles was calculated using the Debye−Scherrer equation, , where is the crystallite size, is the shape factor (0.9), is the X-ray wavelength (1.5406 Å), is the full width at half maximum (FWHM) of the most intense diffraction peak, and is the Bragg angle. Morphology and size distribution of nanoparticles were analysed using a JEOL JEM-2100 TEM at 200 kV. Samples were prepared by dispersing nanoparticles in ethanol and placing a drop onto carbon-coated copper grids. Particle size distribution was calculated from TEM images using ImageJ software.

Mosquito colony maintenance

Anopheles stephensi colonies were maintained in the insectary under controlled conditions of 27 ± 2 °C temperature, 70 ± 5% relative humidity, and a 12:12 h light: dark photoperiod. Larvae were reared in enamel trays filled with dechlorinated tap water and fed a mixture of powdered dog biscuit and yeast in a 3:1 ratio. Emerging adults were kept in mesh cages and provided with 10% sucrose solution ad libitum. Female mosquitoes were blood-fed on restrained rabbits to stimulate egg production, following institutional ethical guidelines for the use of animals.

Larvicidal activity assay

Twenty healthy 2nd and 3rd instar larvae were placed in 250 mL glass beakers containing 100 mL of the test solutions. Five replicates were maintained for each concentration, with controls containing only dechlorinated water. The larvae were not fed during the 24 h exposure period. Mortality was recorded after 24 h by gently probing the larvae to confirm non-responsiveness. Corrected mortality was calculated using Abbott’s formula if control mortality ranged from 5 to 20%, and lethal concentrations (LC50 and LC90) were estimated through probit analysis using SPSS software²¹.

Adult longevity assay

For adult longevity testing, newly emerged 3–5−day−old males and females were separated into treatment groups and provided with cotton pads soaked in 10% sucrose solution containing varying concentrations of curcumin (50–250 µg/mL) or PEG–Beta–Cur–ZnO NPs (25–125 µg/mL). Control groups received only 10% sucrose solution. Cotton pads were replaced daily to ensure freshness. Three replicates, each containing 25 males and 25 females, were maintained for each treatment. Daily mortality was recorded until all individuals had died. Mean longevity was calculated separately for males and females.

Fecundity assay

Fecundity was assessed by transferring blood-fed females from each treatment group to oviposition cups lined with moist filter paper. After 48 h, the filter papers were removed, and the number of eggs laid was counted under a stereomicroscope. Mean fecundity, expressed as the average number of eggs per female, was calculated.

Histopathological examination

For histopathology, 3rd instar larvae exposed to LC50 concentrations of curcumin or PEG–Beta–Cur–ZnO NPs were rinsed with phosphate-buffered saline and fixed in Bouin’s solution for 24 h. The fixed samples were washed with 70% ethanol to remove excess fixative, dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin wax.

Sections of 5 μm thickness were cut using a rotary microtome, mounted on glass slides, deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E). Prepared slides were examined under a light microscope to evaluate structural changes in midgut epithelial tissue, and representative images were captured for documentation.

Bacterial strains and culture conditions

Escherichia coli (MTCC No. 443) and Staphylococcus aureus (MTCC No. 740) were selected as model bacterial strains. Both were cultured overnight in the nutrient broth at 37 °C with constant shaking. The cultures were adjusted to a standard optical density (OD600 ~ 0.1) before all assays to ensure consistent bacterial loads.

Agar well diffusion assay

The antibacterial activity was assessed using the agar well diffusion method. Mueller-Hinton agar plates were uniformly inoculated with the prepared bacterial suspensions. Wells of 6 mm diameter were aseptically punched into the agar and filled with 50 µL of each treatment solution at 50 µg/mL. Plates were incubated at 37 °C for 24 h. The diameter of inhibition zones was measured in millimetres using a digital caliper^22,23.

Live/dead bacterial viability staining

Bacterial viability after treatment was assessed using the Live/Dead BacLight bacterial viability kit. Cultures were treated with curcumin, ZnO NPs, and PEG–Beta–Cur–ZnO NPs at 50 µg/mL for 4 h at 37 °C. Cells were collected, washed with phosphate-buffered saline (PBS), and stained with SYTO 9 and propidium iodide following the manufacturer’s protocol. Samples were visualized under a fluorescence microscope.

Reactive oxygen species (ROS) generation assay

Bacterial suspensions were treated with the formulations (50 µg/mL) for 4 h and then incubated with 2’,7’-dichlorofluorescein diacetate (DCFH-DA, 10 µM) for 30 min in the dark. Cells were washed with PBS, and green fluorescence intensity, indicative of ROS levels, was observed using a fluorescence microscope or quantified with a microplate reader.

Cancer cell culture

The HeLa cervical cancer cell line was sourced from India’s National Centre for Cell Science (NCCS), Pune. HeLa cervical cancer cells were used to evaluate the anticancer activity of curcumin, ZnO NPs, and PEG–Beta–Cur–ZnO NPs. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and maintained at 37 °C in a humidified atmosphere with 5% CO₂.

Assessment of ROS production

HeLa cells were seeded in 24−well plates at 1 × 10⁵ cells per well and incubated overnight at 37 °C in a humidified atmosphere with 5% CO₂ to allow cell attachment. Then the cells were treated with curcumin, ZnO NPs, and PEG–Beta–Cur–ZnO NPs for 24 h. After treatment, the medium was removed, and cells were gently washed twice with phosphate-buffered saline (PBS). A staining solution containing 10 µM DCFH-DA in serum-free medium was added, and cells were incubated for 30 min at 37 °C in the dark to avoid photo-oxidation. Following incubation, excess dye was removed by washing with PBS, and fluorescence images were captured using a fluorescence microscope equipped with appropriate filters (excitation ~ 488 nm, emission ~ 525 nm) to detect ROS-generated green fluorescence.

Live/dead staining

For viability assessment, cells (both treated and control) were washed with PBS and stained with a mixture of calcein−AM (2 µM) and propidium iodide (PI, 4 µM) prepared according to the manufacturer’s protocol. The cells were incubated with the staining solution for 15–30 min at room temperature in the dark. After incubation, cells were washed with PBS to remove unbound dye, and images were acquired using a fluorescence microscope. Live cells exhibited green fluorescence due to calcein−AM, while dead cells stained red with PI.

Assessment of DNA damage

To evaluate nuclear morphology and DNA damage, treated cells were washed with PBS and stained with Hoechst 33,342 dye at a final concentration of 1 µg/mL for 15 min at 37 °C in the dark. Excess dye was removed by PBS washing, and nuclear morphology was observed under a fluorescence microscope using DAPI filters.

Assessment of mitochondrial membrane potential (MMP)

For MMP analysis, cells were washed twice with PBS following treatment and then incubated with JC-1 dye solution (5 µg/mL in serum-free medium) for 30 min at 37 °C in the dark. After staining, cells were gently washed with PBS to remove excess dye and imaged using a fluorescence microscope.

Biochemical assays: SOD, MDA, LDH, and catalase

Hela cells were seeded in 6-well plates at a density of 1 × 10⁵ cells/well and treated with PEG–Beta–Cur–ZnO for 24 h. untreated cells served as controls. After treatment, cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed with RIPA buffer containing protease inhibitors. Lysates were centrifuged at 12,000 × g for 10 min at 4 °C, and the supernatants were collected. Protein concentrations were determined using the Bradford assay. Superoxide dismutase (SOD) activity was measured using a commercial kit based on the inhibition of nitroblue tetrazolium reduction, and absorbance was recorded at 560 nm. Lipid peroxidation was assessed by measuring malondialdehyde (MDA) levels using the thiobarbituric acid reactive substances (TBARS) assay at 532 nm. Lactate dehydrogenase (LDH) release in culture supernatants was determined using a colorimetric kit at 490 nm and expressed as percentage release relative to control. Catalase activity was quantified by measuring the decomposition of hydrogen peroxide at 240 nm.

Caspase-3 and Caspase-9 activity assays

Caspase-3 and caspase-9 activities in Hela cells treated with PEG–Beta–Cur–ZnO, 24 h) were measured using a commercial colorimetric assay kit. The assay is based on cleavage of synthetic tetrapeptide substrates DEVD-pNA (caspase-3) and LEHD-pNA (caspase-9) by active caspases, releasing p-nitroaniline (pNA). Reaction mixtures contained 50 µL of cell lysate protein (50 µg), 50 µL of 2× reaction buffer with 10 mM dithiothreitol, and 5 µL of 4 mM substrate in a total volume of 105 µL. Mixtures were incubated at 37 °C for 1 h, and absorbance of pNA was measured at 405 nm using a microplate reader. Enzyme activities were expressed as picomoles of pNA released per minute per milligram of protein.

Statistical analysis

All experiments were conducted in triplicate, and results were expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism software. One-way ANOVA followed by Tukey’s post-hoc test was used to determine significant differences among groups, with p < 0.05 considered statistically significant. Significance levels were denoted as: **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, and ns = non-significant.

Results and discussion

Characterization

The ZnO–Cur–PEG–β-CD nanocomposite exhibits strong synergistic interactions through hydrogen bonding and electrostatic attraction. β-CD encapsulates Cur within its hydrophobic cavity, enhancing solubility and stability²⁴. PEG acts as a stabilizing and biocompatible linker, improving dispersibility and reducing nanoparticle aggregation. ZnO nanoparticles serve as the core, providing structural integrity and therapeutic activity. Overall, the integrated system enhances curcumin’s bioavailability and ZnO’s biomedical efficacy.

The UV–Vis spectra of ZnO NPs formulations revealed clear evidence of surface modification and stability characteristics (Fig. 1a). Uncoated ZnO NPs displayed an absorption peak at 419 nm, which is inconsistent with another study that stated that ZnO NPs exhibited a distinctive broad absorption peak between 330 and 460 nm²⁵. While PEG–Beta–Cur–ZnO NPs showed a slight shift to 421 nm, confirming successful PEGylation. Further loading with curcumin caused an additional shift to 423 nm, indicating effective drug loading on functionalized NPs that altered the optical properties (Fig. 1a). The observed shifts align with previous reports indicating organic ligands alter ZnO’s optical properties by modifying surface plasmon resonance and band-gap transitions²⁶.

Fig. 1.

a UV-Visible absorption spectra of PEG–Beta–Cur–ZnO nanoparticles, PEG–Beta–ZnO, and ZnO nanoparticles, illustrating characteristic absorption peaks and optical properties. b Stability assessment of PEG–Beta–Cur–ZnO nanoparticles at different time intervals. c Effect of different pH conditions on PEG–Beta–Cur–ZnO nanoparticles.

Stability assessments of PEG–Beta–Cur–ZnO NPs over time (0–180 min) showed a gradual decrease in absorbance intensity, particularly after 90 min, suggesting reduced stability due to aggregation or degradation of the coating (Fig. 1b). This indicates reasonable stability for up to approximately 90 min before significant changes occur. Additionally, pH-dependent stability was evaluated across a range from 2.5 to 10.5. At acidic pH values (2.5 and 4.5), the nanoparticles maintained strong, well-defined absorbance peaks, indicating high stability and minimal aggregation. In contrast, neutral to mildly basic conditions (6.5 and 8.5) led to decreased peak intensity, while at pH 10.5, pronounced absorbance reduction and peak broadening suggested significant aggregation/coating degradation (Fig. 1c). Overall, these findings demonstrate successful PEG–Beta–Cur–ZnO NPs, time-limited stability in solution, and enhanced stability under acidic conditions due to pH-sensitive behavior.

The FTIR spectrum exhibited well-defined absorption bands indicative of functional groups associated with PEG–Beta–Cur–ZnO nanoparticles (Fig. 2a). The broad peak around 3442 cm⁻¹ corresponds to O–H stretching vibrations, confirming the presence of hydroxyl groups contributed by ZnO, PEG, β-cyclodextrin, and curcumin^27,28. Band around 2353 cm⁻¹ was noted, which is attributed to the asymmetric stretching vibration of atmospheric CO₂, a common background feature in FTIR spectra and not related to the chemical structure of the synthesized material. A distinct band observed at 2105 cm⁻¹ corresponds to the stretching vibration of C≡C (alkyne) or C≡N (nitrile) groups, suggesting the possible incorporation of unsaturated or nitrogen-containing moieties within the sample. While the peak at 1640 cm⁻¹ corresponds to C=O stretching vibrations from curcumin²⁹ Additional peaks at 1241, 1037, and 586 cm⁻¹ indicate C–O, C–H bending, and Zn–O stretching vibrations, respectively^30,31. These results confirm the successful conjugation of the PEG–Beta–Cur–NPs surface.

Fig. 2.

a FTIR spectrum of PEG–Beta–Cur–ZnO NPs; b XRD pattern of the nanoparticles; c TEM image illustrating the morphology of the synthesized nanoparticles; d Particle size distribution profile of the nanoparticles based on TEM analysis, e SEM images of PEG–Beta–Cur–ZnO NPs, f The zeta potential of PEG–Beta–Cur–ZnO NPs.

XRD analysis demonstrated sharp and well-defined peaks at 2θ values of approximately 31.8°, 34.4°, 36.3°, 47.5°, 56.6°, and 62.9°, corresponding to the (100), (002), (101), (102), (110), and (103) planes of the hexagonal wurtzite structure of PEG–Beta–Cur–ZnO NPs (Fig. 2b). These results align with a previous study³². The average crystallite size, estimated using the Debye–Scherrer equation, was found to be 65.74 nm. The absence of any additional peaks indicates phase purity and high crystallinity of the nanoparticles, suggesting that the functionalization process did not alter the core crystalline nature of PEG–Beta–Cur–ZnO NPs. XRD results preserved the hexagonal wurtzite structure, indicating that the functionalization process-maintained phase purity, supporting prior findings that surface modifications generally do not disrupt ZnO’s core crystalline lattice^33,34.

TEM images revealed that the nanoparticles were quasi-spherical with a tendency to form aggregates. The morphology shows irregular clustering of particles, likely due to surface interactions between PEG, curcumin, and ZnO (Fig. 2c). Despite the agglomeration, individual nanoparticle boundaries are discernible, supporting the presence of a nanoscale structure. The particle size distribution curve showed a relatively narrow size range, with the majority of particles centred around 76 nm (Fig. 2d). This distribution confirms that the nanoparticles are within the expected nanoscale range and are suitable for biomedical applications where size uniformity is important. Similar size distributions have been reported for PEG-coated ZnO NPs, with PEGylation helping to control particle size and reduce opsonization³⁵. SEM imaging revealed that the PEG–Beta–Cur–ZnO nanoparticles possessed a predominantly spherical morphology with uniform distribution and no visible aggregation (Fig. 2e). The surface appeared slightly rough, indicating successful PEG–β-cyclodextrin and curcumin functionalization. The zeta potential of PEG–Beta–Cur–ZnO NPs was found to be − 38.4 mV, confirming good colloidal stability. The negative surface charge reflects successful PEG–β-cyclodextrin coating and curcumin loading, which enhances nanoparticle dispersion in aqueous media (Fig. 2f).

Anti-malarial vector activity

The larval toxicity assessment demonstrates the effectiveness of curcumin and PEG–Beta–Cur–ZnO NPs against 2nd and 3rd instar larvae of Anopheles stephensi. Curcumin alone shows a concentration-dependent increase in mortality, with higher mortality observed at 200 and 250 µg/mL for both larval stages, indicating moderate larvicidal activity (Fig. 3a, b; Table 1). In contrast, PEG–Beta–Cur–ZnO NPs exhibit enhanced toxicity even at lower concentrations (25–125 µg/mL), achieving significantly higher mortality rates at lower doses compared to free curcumin (Fig. 3a, b; Table 1). Notably, near-complete mortality is observed at 125 µg/mL for both instars, suggesting improved efficacy due to nanoparticle encapsulation and delivery. These results highlight that PEG–Beta–Cur–ZnO NPs significantly enhance the bioavailability and larvicidal potency of curcumin. The results are in agreement with those of Bharathi and Suseem³⁶, who reported that ZnO nanoparticles were highly effective against Anopheles stephensi and Aedes aegypti larvae, demonstrating higher mortality rates after 24 h of exposure compared to CuO nanoparticles.

Fig. 3.

a Larval toxicity of curcumin against 2^nd and 3^rd instar larvae of Anopheles stephensi. b Larval toxicity of PEG–β-Cur–ZnO nanoparticles against 2^nd and 3^rd instar larvae of Anopheles stephensi. Data are presented as mean ± SE, and statistical significance was determined using ANOVA followed by Tukey’s post-hoc test.

Table 1.

Larvicidal effect of Curcumin and PEG–Beta–Cur–ZnO nanoparticles against Anopheles stephensi.

Treatments	Target	LC50 (LC90) (µg/mL)	95% confidence Limit		Regression equation	χ2 (df = 3)
			LC50 (LC90)
			LCL	UCL
Curcumin	2nd Instar	106.797 (260.975)	88.756 (236.115)	121.662 (297.220)	y = 0.888 + 0.008x	2.49 n.s.
	3rd Instar	124.895 (284.508)	108.319 (256.497)	139.520 (325.992)	y = 1.003 + 0.008x	2 59 n.s.
PEG–Beta–Cur–ZnO NPs	2nd Instar	33.189 (114.278)	24.242 (104.972)	40.050 (127.001)	y = 0.777 + 0.023x	3.23 n.s.
	3rd Instar	45.641 (123.174)	35.386 (111.220)	53.614 (140.707)	y = 0.754 + 0.017x	0.32 n.s.

Values are presented as mean ± SD from five independent replicates. LC₅₀ and LC₉₀ represent the lethal concentrations required to cause 50% and 90% larval mortality after 24 h of exposure. Mortality data were analyzed using probit regression analysis. χ² values indicate the goodness of fit of the probit model. Different letters (a, b, c) indicate statistically significant differences between treatments (ANOVA, Tukey test, p < 0.05).

Figure 4a–d, illustrates the effect of curcumin and PEG–Beta–Cur–ZnO–ZnO NPs on the longevity and fecundity of Anopheles stephensi. Curcumin treatment shows a clear dose-dependent reduction in adult lifespan for both males and females, with significant decreases at concentrations of 50–250 µg/mL. Male longevity declines from around 33 days in controls to about 22.5 days at the highest concentration, while females show an even sharper reduction from approximately 33 days to about 18.5 days. In addition, fecundity analysis revealed a significant reduction in egg production with increasing concentrations. The average number of eggs laid dropped from over 220 eggs per female in the control to around 122 eggs at 250 µg/mL, indicating the reproductive toxicity of the nanoparticle formulation.

Fig. 4.

a, b Bar graphs representing the longevity and fecundity of male and female Anopheles stephensi after treatment with Curcumin, highlighting its impact on lifespan and reproductive potential. c,d Bar graphs illustrating the effects of exposure to Curcumin-loaded β-cyclodextrin-coated ZnO nanoparticles (PEG-Beta–Cur-ZnO NPs) on the longevity and fecundity of Anopheles stephensi, demonstrating potential reductions in survival and reproductive output. Tukey’s HSD test (α = 0.05) was used, where values sharing the same letter indicate no significant difference. Standard deviations are depicted as T-bars. Data represent the mean ± standard deviation from five replicates. Statistical significance was set at p < 0.05. Significance levels are denoted as **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, and ns (non-significant). One-way and two-way ANOVA were used for statistical comparisons between groups.

In comparison, PEG–Beta–Cur–ZnO NPs demonstrate even stronger effects at lower doses (25–125 µg/mL). Both male and female adult longevity is significantly reduced, with males declining from 35days to approximately 21.2 days and females showing a similar trend. Fecundity results highlight a dramatic reduction in reproductive output, with egg production falling from around 250 in controls to just over 100.5 eggs at 125 µg/mL. The nanoparticle formulation is notably more effective than free curcumin, achieving greater mortality and reproductive suppression at lower concentrations. Green-synthesized ZnO nanoparticles were reported to reduce adult longevity and fecundity in Anopheles species³⁷. These results are consistent with the present findings and are further supported by Sureh et al.³⁸, who observed that algae-synthesized carbon quantum dots enhanced adult mortality and suppressed reproductive capacity in insect vectors.

Histopathological analysis of third-instar Anopheles stephensi larvae reveals significant differences in midgut tissue architecture across treatment groups (Fig. 5). In the control larvae, the midgut shows a well-defined epithelial cell lining with intact cellular structures and organized tissue morphology, indicating normal physiological conditions. Larvae treated with curcumin exhibit moderate histological changes, including partial disruption of the epithelial layer and signs of cellular degeneration. The tissue structure appears less compact, with visible vacuolation and mild disorganization, suggesting that curcumin induces some level of physiological stress and damage to the midgut. In contrast, larvae exposed to PEG–Beta–Cur–ZnO NPs show pronounced histopathological alterations. The midgut epithelium is severely damaged, with extensive vacuolation, cell lysis, and disrupted structural integrity. The cellular disorganization and breakdown of tissue layers suggest that the nanoparticle formulation causes substantial internal damage due to enhanced bioavailability and deeper penetration into larval tissues. The present findings align with those of Kalpana et al.³⁹, who demonstrated that zinc oxide nanoparticles synthesized from U. lactuca caused severe damage to the cuticular surface of Anopheles stephensilarvae, along with noticeable zinc accumulation inside the larval tissues. Similarly, Suresh et al.⁴⁰, reported that treatment with cerium doped silver nanoparticles produced pronounced histopathological modifications in the midgut and digestive organs of third-instar A. aegypti larvae, emphasizing the harmful influence of such nanomaterials.

Fig. 5.

Histopathological profiles of third-instar Anopheles stephensi larvae under different treatment conditions. The images show the structural integrity of a control larvae, b larvae treated with Curcumin, and c larvae exposed to PEG-Beta–Cur-ZnO nanoparticles (PEG–Beta–Cur–ZnO NPs). Magnification: ×100.

Antibacterial activity

The antibacterial activity of PEG–Beta–Cur–ZnO nanoparticles was assessed against Escherichia coli and Streptococcus aureus by determining the diameter of the inhibition zones using the agar well diffusion method (Fig. 6). For Escherichia coli, curcumin exhibited a modest inhibition zone of 7.3 ± 0.05 mm, while ZnO NPs showed a larger zone of 11.5 ± 0.25 mm. PEG–Beta–Cur–ZnO NPs demonstrated enhanced antibacterial activity with a zone of inhibition measuring 13.4 ± 0.17 mm. Against Streptococcus aureus, curcumin showed an inhibition zone of 8.4 ± 0.10 mm. ZnO NPs exhibited a greater effect with 13.5 ± 0.05 mm, while PEG–Beta–Cur–ZnO NPs resulted in a further increased zone of 15.2 ± 0.05 mm. These results indicate that PEG–Beta–Cur–ZnO NPs possess superior antibacterial efficacy compared to unmodified ZnO NPs and curcumin alone against both bacterial strains tested. Nanoformulated drugs show higher potency by enhancing cellular uptake and ensuring sustained release, maintaining effective concentrations. They also combine the antibiotic’s action with the intrinsic antimicrobial effects of ZnO nanoparticles. This synergy disrupts bacterial membranes and biofilms more effectively than the free drug⁴¹. Ibne Shoukani et al⁴². reported that the nanoformulated ciprofloxacin conjugated and coated with PEG (CIP–PEG–ZnO–NPs) exhibited a significantly larger zone of inhibition against Salmonella typhi, Staphylococcus aureus, MRSA, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa compared to the free ciprofloxacin drug.

Fig. 6.

Antibacterial activity against Escherichia coli and Staphylococcus aureus following treatment with curcumin, ZnO nanoparticles, and PEG–Beta–Cur–ZnO nanoparticles. All bacterial cultures were exposed to a standardized concentration of 50 µg/ML. (C-Control, (1) Curcumin (2) ZnO NPs, (3) PEG–Beta–Cur–ZnO NPs.

Nanoparticles exhibit potent antibacterial effects through apoptosis−like mechanisms. In this present study, the apoptotic effect of PEG–Beta–Cur–ZnO NPs was evaluated using live/dead staining (Fig. 7). Untreated control bacterial cells show predominantly green fluorescence, indicating healthy, viable cells. Curcumin treatment caused a slight increase in red fluorescence, suggesting mild bacterial membrane damage and cell death. ZnO NPs showed stronger antibacterial effects, with increased red fluorescence reflecting disrupted membranes and higher proportions of dead cells in both bacterial species. PEG–Beta–Cur–ZnO NPs NPs exhibited the most intense red fluorescence and widespread membrane damage, indicating the highest bacterial mortality. This enhanced antibacterial activity is attributed to the synergistic effects of curcumin and ZnO, improved cellular uptake, and sustained release enabled by PEGylation. PEG-functionalized graphene oxide nanoparticles loaded with Nigella sativa extract have demonstrated the ability to induce apoptosis in Staphylococcus aureus and Escherichia coli⁴³.

Fig. 7.

Live/dead bacterial cell viability was evaluated in Escherichia coli and Staphylococcus aureus following treatment with curcumin, ZnO nanoparticles, and PEG–Beta–Cur–ZnO nanoparticles. Each formulation was applied at a standardized concentration of 50 µg/mL, and comparisons were made between treated and untreated groups. Statistical analysis was performed using GraphPad Prism, with significance set at p < 0.05; **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, and ns = non-significant.

The effect of PEG–Beta–Cur–ZnO NPs on ROS production in Escherichia coli and Staphylococcus aureus was evaluated using DCFH-DA staining (Fig. 8). Control bacterial cells showed minimal green fluorescence, indicating low ROS levels and healthy cellular states. Curcumin treatment resulted in moderate ROS generation with visible increases in green fluorescence. ZnO NPs produced higher ROS levels, indicating their potential to induce oxidative stress, leading to damage of bacterial membranes and intracellular components. PEG–Beta–Cur–ZnO NPs caused the most intense green fluorescence, indicating a substantial increase in ROS production. This enhanced ROS generation is responsible for the strong antibacterial effects observed with the nanoformulation, reflecting a synergistic mechanism where curcumin and ZnO together promote oxidative stress more effectively. These findings align with previous research^44,45.

Fig. 8.

ROS generation in Escherichia coli and Staphylococcus aureus following treatment with curcumin, ZnO nanoparticles, and PEG–Beta–Cur–ZnO nanoparticles. The analysis highlights the correlation between relative ROS levels and varying concentrations of the different nano formulations. Statistical analysis was performed using GraphPad Prism, with significance set at p < 0.05; **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, and ns = non-significant.

Anticancer activity

The effect of PEG–Beta–Cur–ZnO NPs on ROS production and apoptosis was assessed in HeLa cells by using DCFH-DA and AO/EtBr staining, respectively (Fig. 9). Fluorescence imaging using DCFH-DA dyes revealed minimal ROS generation in control cells. In contrast, curcumin and ZnO NPs caused moderate increases in ROS production, while PEG–Beta–Cur–ZnO NPs induced a substantial rise in fluorescence intensity, indicating pronounced oxidative stress. Live/dead staining further demonstrated the apoptotic effects of PEG–Beta–Cur–ZnO NPs. Control cells remained viable, showing strong green fluorescence. Curcumin and ZnO NPs led to increased red fluorescence, representing elevated cell death with moderate toxicity. PEG–Beta–Cur–ZnO NPs caused a dramatic shift, with intense red fluorescence and reduced green signal, indicating extensive cell death. These findings align with earlier work demonstrating that drug-loaded ZnO NPs and curcumin-loaded nanoparticles induce cytotoxicity through ROS-mediated apoptosis and mitochondrial dysfunction^46–48. Similarly, Dong et al⁴⁹. reported that ZnO@Cur exhibited higher ROS generation and greater apoptotic effects than free curcumin in both A549 and HEL-299 cells.

Fig. 9.

Alterations in ROS levels in HeLa cells following treatment with curcumin, ZnO nanoparticles, and PEG–Beta–Cur–ZnO nanoparticles were evaluated. Cell viability was assessed using a live/dead fluorescence assay after 24 h of exposure. Viable cells stained with Calcein-AM exhibited green fluorescence, whereas non-viable cells stained with propidium iodide (PI) displayed red fluorescence. Quantification of live and dead cells was performed across randomly selected microscopic fields. Data are presented as mean ± standard deviation (SD) from three independent experiments. Statistical significance was determined at p < 0.05 and p < 0.01.

The impact of curcumin, ZnO NPs, and PEG–Beta–Cur–ZnO NPs on DNA damage and MMP was assessed in HeLa cancer cells. Control cells showed minimal fluorescence, indicating stable nuclear integrity and healthy mitochondrial function. Treatment with curcumin and ZnO NPs caused a marked increase in DNA fragmentation, observed as enhanced blue fluorescence in nuclei, and clear mitochondrial membrane depolarization, indicated by stronger orange staining (Fig. 10). These results suggest that both agents induce moderate genotoxic and mitochondrial stress in HeLa cells. The PEG–Beta–Cur–ZnO NPs treated group exhibited the most pronounced effects, with intense nuclear fluorescence signaling extensive DNA damage and strong orange fluorescence revealing severe MMP loss. These results demonstrate that PEG–Beta–Cur–ZnO NPs exert enhanced cytotoxicity against HeLa cancer cells by triggering both nuclear and mitochondrial damage pathways. The synergistic action of curcumin and ZnO in nanoparticle form intensifies these effects, supporting their potential as a promising strategy for anticancer therapy by promoting oxidative stress, genotoxicity, and mitochondrial dysfunction in malignant cells. Yang et al.⁵⁰ reported that, compared to the free drug DOX, the nano formulated ZnO-PG-RGD/DOX induced significant nuclear alterations in U87 cells. Hannachi et al.⁵¹ also reported that ZnO nanoparticles co-doped with yttrium (Y) and cerium (Ce) induced nuclear alterations in HCT-116 colon cancer cells.

Fig. 10.

DNA damage and mitochontrial potantial of Curcumin, ZnO NPs, and PEG–Beta–Cur–ZnO NPs. Statistical analysis was performed using GraphPad Prism, with significance set at p < 0.05; **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, and ns = non-significant.

Exposure of HeLa cells to PEG–Beta–Cur–ZnO nanoparticles for 24 h resulted in pronounced biochemical and enzymatic alterations characteristic of oxidative stress and apoptosis (Fig. 11a–f). A substantial increase in apoptotic enzyme activity was observed across all treatment groups. Caspase-3 activity rose from 0.246 mU/mL in control cells to 1.66, 2.46, and 3.56 mU/mL following Curcumin, ZnO NPs, and PEG-Beta-Cur-ZnO treatments, respectively. Caspase-9 showed a comparable trend, increasing from 0.3 mU/mL in controls to 1.86, 3.06, and 4.16 mU/mL. These elevations confirm activation of the intrinsic mitochondrial apoptotic pathway, consistent with previous reports showing ZnO-NP–induced mitochondrial dysfunction and caspase activation in cancer cells⁵².

Fig. 11.

Caspase-3 activity (A), caspase-9 activity (B), superoxide dismutase (SOD) levels (C), catalase levels (D), malondialdehyde (MDA) content (E), and lactate dehydrogenase (LDH) activity (F) in HeLa cells following treatment with curcumin, ZnO NPs, and PEG–Beta–Cur–ZnO NPs. Data are represented as mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. Significance indicators: **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, and ns = non-significant compared to the control group.

The oxidative stress response mirrored apoptotic activation. Antioxidant enzyme levels increased significantly, indicating a compensatory reaction to elevated ROS. SOD activity increased from 25 U/mL in control cells to 62, 80.66, and 94.33 U/mL, whereas CAT activity rose from 16.33 to 44.33, 61.33, and 79.33 U/mL following the same treatments. Similar antioxidant upregulation in nanoparticle-treated cells has been documented as a cellular defense against oxidative imbalance^53,54. Oxidative damage markers confirmed the elevated ROS burden. MDA, an index of lipid peroxidation, increased from 13.66 µM in controls to 35.66, 45.66, and 61 µM with Curcumin, ZnO NPs, and PEG–Beta–Cur–ZnO, respectively. The highest MDA level observed with PEG–Beta–Cur–ZnO indicates a stronger membrane-damaging effect, consistent with studies demonstrating enhanced lipid peroxidation and cytotoxicity upon functionalized ZnO nanocomposite exposure^55,56. These findings demonstrate that PEG–Beta–Cur–ZnO nanoparticles induce substantial oxidative stress and apoptosis in HeLa cells, surpassing the effects elicited by Curcumin or ZnO NPs alone. The combined elevation of caspase activity, increased levels of SOD and CAT, and intensified lipid peroxidation indicates oxidative-damage–mediated, mitochondria-dependent cell death. These outcomes align with existing literature highlighting enhanced anticancer activity of curcumin-based nanoconjugates due to improved cellular uptake and ROS-triggered apoptotic signalling⁵⁷. Thus, PEG–Beta–Cur–ZnO represents a potent oxidative stress inducer with significant cytotoxic potential in cancer cell systems.

Biocompatibility

The biocompatibility of curcumin, ZnO NPs, and PEG–Beta–Cur–ZnO NPs was evaluated using fibroblast cells. An MTT assay was performed at concentrations up to 125µg/mL. As shown in Fig. 12, all three curcumin, ZnO NPs, and PEG–Beta–Cur–ZnO NPs exhibited excellent biocompatibility, with no detectable cytotoxicity toward fibroblast cells across the tested concentration range. These results indicate that the formulations are safe and non-toxic to normal cells at the evaluated doses.

Fig. 12.

Fibroblast viability was assessed using the MTT assay. No significant cytotoxicity was observed for curcumin, ZnO NPs, or PEG–Beta–Cur–ZnO NPs up to 125 µg/mL.

Conclusion

PEG–Beta–Cur–ZnO NPs were successfully synthesized with confirmed surface functionalization and pH-sensitive stability. NPs demonstrated significantly enhanced bioactivity, showing strong larvicidal effects against Anopheles stephensi, superior antibacterial efficacy against Escherichia coli and Staphylococcus aureus, and potent anticancer activity in HeLa cells through increased ROS generation, DNA damage, and mitochondrial dysfunction (Fig. 13). These results highlight the synergistic advantages of PEG and curcumin functionalization in improving bioavailability and targeted action. Overall, PEG–Beta–Cur–ZnO nanoparticles represent a promising multifunctional platform for eco-friendly vector control, broad-spectrum antibacterial applications, and in vitro anticancer potential, warranting further in vivo and long-term toxicity studies to confirm their clinical applicability.

Fig. 13.

Schematic illustration of PEG–Beta–Cur–ZnO NPs, their characterization, and multifunctional biological activities including anti-malarial, antibacterial, and anticancer effects through ROS generation, oxidative stress, and apoptosis mechanisms.

Author contributions

Conceptualization was carried out by US, CP, MMH and ATA. Data curation was handled by MS, ZMM and AS. Methodology was developed by US, CP, and ATA. The original draft was written by CP, MS, MMH and AA, while the review and editing were performed by ZMM and CP. All authors have read and agreed to the published version of the manuscript.

Funding

This article is derived from a research grant funded by the Research, Development, and Innovation Authority (RDIA) - Kingdom of Saudi Arabia with grant number (13445-Tabuk-2023-UT-R-3-1-SE). Also, the authors would like to express their appreciation to the Deanship of Scientific Research, University of Tabuk, Kingdom of Saudi Arabia for funding this research under the project number S-0285-1440.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.