Targeted Folic Acid‑Conjugated Chitosan Nanoparticles Deliver Genistein, Boosting Anticancer Efficacy Against Cervical Cancer

Abstract

In this work, we engineered folic acid–conjugated chitosan nanoparticles (FGCN) to deliver the natural isoflavone genistein (GEN) specifically to cervical cancer cells. Compared with plain GEN‑loaded chitosan nanoparticles (GCN), the FGCN exhibited a slightly larger hydrodynamic diameter (~165 nm) and a modestly reduced surface charge (+21.5 mV). Both systems achieved >95 % drug entrapment and controlled release in phosphate‑buffered saline (pH 7.4), releasing ~90 % of GEN over 24 h. Cellular uptake studies in HeLa cells showed a statistically significant (p < 0.05) 2–3‑fold increase in internalization for FGCN versus GCN, attributable to the high affinity of folic acid for the overexpressed folate receptor‑α (FR‑α). Cytotoxicity assays revealed that the IC₅₀ of GEN dropped from 33.8 µg/mL (free drug) to 14.6 µg/mL when delivered via FGCN, while GCN reduced it to 26.5 µg/mL. Apoptosis analysis demonstrated that FGCN induced >55 % apoptotic cell death, markedly higher than GCN (~22 %) and free GEN. These findings confirm that folate conjugation enhances GEN delivery, cellular uptake, and antitumor efficacy in cervical cancer cells.

Background

Cervical cancer remains a leading cause of morbidity among women worldwide, primarily driven by high‑risk human papillomavirus (HPV) infection. While prophylactic vaccines (Gardasil, Cervarix) and conventional therapies—surgery, radiotherapy, and chemotherapy—are available, they are insufficient for many patients, especially in low‑resource settings. Natural compounds such as flavonoids offer anticancer activity with lower systemic toxicity. Genistein, a soy isoflavone, inhibits protein tyrosine kinases and NF‑κB, arrests the cell cycle at G2/M, and induces apoptosis; however, its clinical utility is limited by poor aqueous solubility (~1.45 µg/mL) and low bioavailability. Nanoparticle encapsulation can improve these physicochemical properties, enhance tumor accumulation via the enhanced permeation and retention (EPR) effect, and overcome multidrug resistance.

Chitosan, a biodegradable polysaccharide rich in primary amines, is biocompatible and amenable to surface modification. Conjugating folic acid (FA) to chitosan targets the FA receptor‑α, overexpressed on many cervical cancer cells, enabling receptor‑mediated endocytosis and selective delivery.

Methods

Materials

Genistein (Aladdin Chemicals, Shanghai), chitosan (85 % deacetylated, Sigma‑Aldrich), folic acid, EDC, and NHS (Sigma‑Aldrich) were used as received.

Preparation of GEN‑loaded FA‑conjugated Chitosan Nanoparticles

Folic acid was first activated with EDC/NHS in DMSO and conjugated to chitosan (0.5 % w/w) under stirring at pH 8. The resulting FA‑chitosan conjugate was purified by dialysis. GEN, chitosan, and FA‑chitosan (10:1 w/w) were dissolved in DMSO and added dropwise to 0.5 % Tween‑80, followed by stirring for 3 h. The solvent was evaporated, and the suspension was centrifuged at 10 000 rpm (4 °C) for 30 min. Drug loading (DL%) and entrapment efficiency (EE%) were calculated by HPLC (Agilent LC‑1100, C18 column, 60 % MeOH/40 % H₂O, 1 mL min⁻¹).

Particle Size and Surface Morphology

Dynamic light scattering (Malvern Zetasizer Nano ZS90) measured size and polydispersity. Transmission electron microscopy (JEOL JEM‑1230, 100 kV) visualized morphology.

In Vitro Drug Release

Release was assessed by dialysis (MWCO 3500) in PBS (pH 7.4) at 37 °C with gentle stirring. Samples were collected at predetermined intervals and analyzed by HPLC.

Cell Culture and Uptake

HeLa cells (ATCC) were maintained in DMEM +10 % FBS. Cellular uptake of coumarin‑6–loaded GCN and FGCN was quantified by fluorescence microplate reading (excitation 430 nm, emission 485 nm) and visualized by confocal laser scanning microscopy (Olympus Fluoview).

Cytotoxicity, Live/Dead, and Apoptosis Assays

MTT (5 mg mL⁻¹) measured viability; IC₅₀ values were fitted using GraphPad Prism. Live/dead staining employed calcein AM/ethidium homodimer. Apoptosis was quantified by annexin V/PI staining and flow cytometry.

Statistical Analysis

Data are presented as mean ± SD (n = 3). Statistical significance was evaluated by Student’s t‑test (p < 0.05).

Results and Discussion

Physicochemical Characterization

GCN exhibited an average diameter of 140 nm (+26 mV), while FGCN measured 165 nm (+21.5 mV). Both had EE% >95 % and DL% within 10–12 %. Size and zeta potential remained stable after 3 months storage at 4 °C.

In Vitro Release Kinetics

Both GCN and FGCN released ~35–40 % of GEN within the first 4 h, achieving ~90 % cumulative release over 24 h. The slight delay in FGCN release is attributed to the FA coating, which prolongs drug diffusion.

Cellular Uptake

Fluorescence assays showed a 2–3‑fold higher uptake of FGCN compared with GCN at 4 h (p < 0.05). Confocal images confirmed intense intracellular fluorescence for FGCN, indicating successful FA‑mediated endocytosis.

Cytotoxicity

IC₅₀ values: free GEN 33.8 µg mL⁻¹ → GCN 26.5 µg mL⁻¹ → FGCN 14.6 µg mL⁻¹ (24 h). The superior efficacy of FGCN is linked to enhanced uptake and controlled release, reducing P‑glycoprotein–mediated efflux.

Apoptosis

Annexin V/PI staining revealed apoptosis rates of 0 % (free GEN), 22 % (GCN), and 55 % (FGCN), underscoring the potency of targeted delivery.

Live/Dead Assay

FGCN-treated cells displayed minimal green fluorescence and abundant red staining, consistent with high cell death, whereas free GEN-treated cells remained largely viable.

Conclusions

FA‑conjugated chitosan nanoparticles markedly improve the delivery of genistein to HeLa cervical cancer cells, enhancing cellular uptake, lowering IC₅₀ to 14.6 µg mL⁻¹, and inducing >55 % apoptosis. These results support further preclinical evaluation of FGCN as a promising targeted anticancer platform.

Abbreviations

EPR

Enhanced permeation and retention

FGCN

Folic acid‑conjugated GEN‑loaded chitosan nanoparticles

FR

Folate receptor

GCN

GEN‑loaded chitosan nanoparticles

GEN

Genistein