Optimized Mitoxantrone Delivery Using Cholesterol‑Modified Pullulan Nanoparticles: Size‑Dependent Antitumor Efficacy Against Bladder Cancer

Abstract

Enhancing chemotherapeutic delivery with nanoparticles can lower systemic doses and reduce toxicity. In this study we engineered cholesterol‑substituted pullulan polymers (CHPs) to load mitoxantrone and systematically varied the hydrophobic substitution to generate nanoparticles (NPs) of distinct sizes. Three CHPs—CHP‑1 (6.82 % cholesterol, 86.4 nm), CHP‑2 (5.78 % cholesterol, 162.3 nm) and CHP‑3 (2.74 % cholesterol, 222.3 nm)—were characterized by ^1H NMR and dynamic light scattering. Mitoxantrone release in phosphate‑buffered saline (PBS, pH 7.4) over 48 h reached 38.7 %, 42.4 % and 58.9 % for CHP‑1, CHP‑2 and CHP‑3, respectively. Acidic media (pH 4.0) accelerated release, particularly for the largest particles (75.5 % after 48 h). In vitro, CHP‑3 NPs exhibited the strongest cytotoxicity (IC_50 = 0.25 µM at 24 h) and induced the highest apoptosis and migration inhibition in MB49 bladder‑cancer cells. These results demonstrate that larger, cholesterol‑rich CHPs enhance drug loading, sustain release, and improve antitumor activity.

Background

Traditional chemotherapy suffers from non‑specific distribution and severe side effects, notably cardiotoxicity of anthracyclines such as mitoxantrone. Nanoparticle‑based carriers can exploit the enhanced permeability and retention (EPR) effect to accumulate preferentially in tumor tissue, thereby improving therapeutic index. Pullulan, a non‑toxic, biodegradable polysaccharide, can be rendered amphiphilic by grafting hydrophobic cholesterol via a succinic spacer, yielding self‑assembling nanospheres with a hydrophobic core for drug encapsulation.

Nanoparticle size critically influences cellular uptake, drug release, and biodistribution. Optimal endocytosis typically occurs for particles in the 25–50 nm range, while larger particles may persist longer in circulation and release payloads more gradually. The present work investigates how varying cholesterol substitution—and thus NP size—modulates mitoxantrone release and antitumor efficacy.

Materials and Methods

Reagents

Mitoxantrone (Aladdin Chemistry), pullulan, succinic anhydride cholesterol ester (CHS), and other reagents were purchased from standard suppliers. Dialysis membranes (MWCO 8–12 kDa) were used for drug loading.

CHP Synthesis and Characterization

Pullulan (0.5 g) was reacted with CHS (feed ratios 0.20, 0.15, 0.05 mmol mmol^−1) in DMSO under activation with DMAP and EDC. The resulting CHPs were precipitated in ethanol, purified, and dried. ^1H NMR (DMSO‑d_6) confirmed cholesterol grafting and quantified the degree of substitution (DS) using the methylene peak at 2.53 ppm. DS values were 6.82 % (CHP‑1), 5.78 % (CHP‑2) and 2.74 % (CHP‑3).

Drug‑Loaded NP Preparation

Mitoxantrone (4 mg) was incorporated into each CHP (40 mg) by dialysis against PBS. The final NPs were lyophilized, redispersed in water, and analyzed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Zeta potential was −1.12 mV.

In Vitro Release

NPs were incubated in PBS (pH 7.4), acetate buffer (pH 6.8) and citrate buffer (pH 4.0) at 37 °C. Drug release was quantified by UV–vis spectrophotometry at 490 nm, and cumulative release percentages (Q%) were calculated over 48 h.

Cell Studies

Murine bladder‑cancer MB49 cells were cultured in DMEM + 10 % FBS. Cytotoxicity was measured by MTT at 24, 48 and 72 h; IC_50 values were derived from dose–response curves. Apoptosis was assessed via Annexin V/PI staining and flow cytometry. Cell migration was evaluated using a scratch‑wound assay, with image analysis performed by ImageJ.

Results and Discussion

CHP Structural Confirmation

^1H NMR spectra displayed characteristic pullulan signals and new peaks (0.40–2.40 ppm) confirming cholesterol grafting. The calculated DS matched the intended feed ratios, confirming controlled substitution.

NP Size and Morphology

Blank CHPs formed nanoparticles of 79.1, 104.9 and 166.8 nm for CHP‑1, CHP‑2 and CHP‑3, respectively. Upon drug loading, sizes increased to 86.4, 162.3 and 222.3 nm, reflecting drug encapsulation within the hydrophobic core. TEM images revealed uniform, spherical particles.

Drug Loading and Release

Loading efficiencies were 88.9 % (CHP‑1), 82.3 % (CHP‑2) and 50.7 % (CHP‑3). Release profiles showed sustained release in neutral pH, with larger particles releasing more drug over 48 h. Acidic conditions accelerated release: at pH 4.0, cumulative release reached 75.5 % for CHP‑3.

Antitumor Activity

MTT assays revealed IC_50 values of 0.25 µM (CHP‑3) at 24 h, compared to 0.20 µM (CHP‑2) and 0.06 µM (free mitoxantrone). Apoptosis assays confirmed higher early and late apoptotic rates for CHP‑3, followed by CHP‑2 and CHP‑1. Migration assays showed comparable inhibition by free drug and NPs, with CHP‑3 achieving the most pronounced effect.

Mechanistic Insights

The superior efficacy of larger CHPs likely stems from enhanced extracellular drug release within the acidic tumor microenvironment, coupled with improved cellular uptake via pinocytosis. The hydrophobic core stability governs release kinetics, while surface charge remains neutral, favoring biodistribution.

Conclusion

Cholesterol‑modified pullulan nanoparticles exhibit size‑dependent drug loading, release, and antitumor activity. Larger, cholesterol‑rich NPs (CHP‑3) provide superior mitoxantrone delivery, achieve higher cytotoxicity and apoptosis, and more effectively inhibit bladder‑cancer cell migration. These findings support further development of CHPs as safe, biodegradable nanocarriers for targeted chemotherapy.