Resveratrol-Loaded Human Serum Albumin Nanoparticles with PEG–RGD Targeting: Extended Circulation, Enhanced Biocompatibility, and Potent Pancreatic Tumor Suppression

Abstract

Human serum albumin (HSA) is a natural, non‑immunogenic carrier that can encapsulate lipophilic drugs and improve their pharmacokinetics. We engineered resveratrol (RV)-laden HSA nanoparticles conjugated with arginine–glycine–aspartate (RGD) via a polyethylene glycol (PEG) linker (HRP–RGD NPs) to achieve targeted delivery to pancreatic tumors. The nanoparticles exhibit a uniform 120 ± 2.6 nm diameter, a 62.5 ± 4.21 % RV encapsulation efficiency, and a pH‑sensitive release of 58.4 ± 2.8 % at pH 5.0 and 37 °C. PEG and HSA coatings markedly improve RV biocompatibility. Confocal imaging shows a 47.3 ± 4.6 % uptake of HRP–RGD NPs in PANC‑1 cells, outperforming non‑targeted controls. In vivo, HRP–RGD NPs extend RV’s half‑life 5.43‑fold, achieving 3.01‑ and 8.1‑fold higher tumor accumulation than HRP NPs and free RV, respectively. Treatment of tumor‑bearing mice with HRP–RGD NPs completely suppressed tumor growth over 35 days, with no systemic toxicity. These data demonstrate that HRP–RGD NPs provide a robust, biocompatible platform for targeted pancreatic cancer therapy.

Background

Pancreatic cancer remains one of the most lethal malignancies, with a median survival of less than six months and a 5‑year survival rate below 6 %. Conventional treatments—surgery, radiotherapy, and conventional chemotherapy—are hampered by severe side effects and limited efficacy. Resveratrol (RV), a natural polyphenol from grapes and soybeans, shows anticancer activity across several tumor types but suffers from poor solubility, rapid clearance, and non‑specific distribution. Encapsulation strategies, particularly using endogenous carriers such as HSA, can enhance drug solubility, extend circulation, and enable functionalization for active targeting. HSA is non‑toxic, non‑immunogenic, and possesses reactive carboxyl and amino groups for conjugation. PEGylation further prolongs blood residence time.

Methods

Materials

All reagents were sourced from Sigma Aldrich, Seebio Biotech, and Pierce Biotech. Human serum albumin (≥96 % purity), resveratrol (≥99 %), and RGD peptide (≥97 %) were used without further purification.

Synthesis of HSA–RV Nanoparticles

Resveratrol (6 mg in 1 mg mL⁻¹ DMSO) was mixed with HSA (10 mg mL⁻¹) and stirred for 6 h at room temperature to form coacervates. Cross‑linking with 0.5 % glutaraldehyde (100 µL) followed by dialysis produced HSA–RV NPs. Blank NPs were prepared identically without RV.

Conjugation of PEG–RGD and Formation of HRP–RGD NPs

PEG‑2000‑COOH was activated with SATA and coupled to RGD in the presence of EDC, forming SATA‑PEG‑RGD. After reduction with hydroxylamine, the product was purified and reacted with SPDP‑activated HSA–RV NPs to yield HRP–RGD NPs. Excess reagents were removed by dialysis and filtration.

Characterization

• Size and polydispersity: Dynamic light scattering (DLS) and SEM.
• Drug loading: UV–Vis spectrophotometry at 306 nm.
• Release kinetics: Dialysis bag (8–12 kDa MWCO) in PBS at pH 5.0, 6.5, 7.4, 9.0, 37 °C.

Biocompatibility and Hemolysis

RBCs were incubated with HRP–RGD NPs (10–200 µg mL⁻¹) for 3 h at 37 °C. Hemolysis ratios were calculated against PBS (negative) and deionized water (positive) controls.

Cellular Studies

PANC‑1 cells were cultured in DMEM with 10 % FBS. FITC‑labelled HRP–RGD NPs were incubated for 5 h, followed by confocal imaging and flow cytometry to assess uptake. Cytotoxicity was evaluated by CCK‑8 assay across 0–200 µg mL⁻¹ concentrations. Apoptotic morphology was confirmed by Hoechst 33258 staining.

Animal Experiments

Balb/c nude mice were injected subcutaneously with 1 × 10⁶ PANC‑1 cells. When tumors reached ~80 mm³, mice were randomized (n = 5) into saline, RV, HRP NPs, or HRP–RGD NPs groups (10 mg kg⁻¹ RV equivalent). Tumor volume and body weight were monitored every 3 days for 35 days. Biodistribution was assessed at 24 h via UV–Vis analysis of tumor tissue. Histopathology of major organs was performed post‑mortem.

Results and Discussion

Nanoparticle Fabrication and Physicochemical Properties

HRP–RGD NPs displayed a narrow, unimodal size distribution (120 ± 2.6 nm) with a homogeneous spherical morphology. UV–Vis spectra confirmed RV encapsulation, while fluorescence emission at 325 nm corroborated RV retention within the particles.

Drug Loading and pH‑Responsive Release

The encapsulation efficiency peaked at 62.5 ± 4.21 %. Release studies showed a 58.4 ± 2.8 % cumulative release after 60 h at pH 5.0, compared to significantly lower rates at physiological pH, highlighting tumor‑specific release.

Enhanced Stability and Biocompatibility

Unlike crystalline free RV, HRP–RGD NPs remained colloidally stable in PBS, DMEM, and FBS over 4 weeks. Fluorescence intensity remained >96 % after 4 weeks, whereas free RV dropped to 12 %. Hemolysis assays revealed <5 % hemolysis up to 200 µg mL⁻¹, matching PBS controls.

Targeted Cellular Uptake

Flow cytometry quantified a 58.5 ± 3.5 % uptake of HRP–RGD NPs by PANC‑1 cells, markedly higher than non‑targeted HRP NPs (16.2 ± 4.9 %) and RGD‑blocked controls (7.1 ± 5.1 %). Confocal imaging corroborated efficient intracellular delivery.

Potent Antitumor Activity In Vitro

HRP–RGD NPs exhibited dose‑dependent cytotoxicity, reducing PANC‑1 viability to <30 % at 50 µg mL⁻¹, outperforming free RV and HRP NPs. Hoechst staining revealed characteristic apoptotic nuclear condensation.

Prolonged Circulation and Tumor Accumulation

HRP–RGD NPs extended RV’s plasma half‑life to 6.57 ± 0.9 h (vs. 1.21 ± 0.09 h for free RV). At 24 h, tumor RV content in the HRP–RGD group was 3.01‑ and 8.1‑fold higher than HRP NPs and free RV, respectively, indicating effective EPR and RGD‑mediated targeting.

In Vivo Tumor Suppression and Safety

HRP–RGD NP treatment completely halted tumor growth over 35 days, with no relapse. Body weights remained stable across all groups. Histology of heart, liver, spleen, lung, and kidney showed no pathological changes, confirming systemic safety.

Conclusions

HRP–RGD NPs represent a clinically relevant platform that overcomes the solubility, pharmacokinetic, and targeting limitations of free resveratrol. By combining HSA encapsulation, PEGylation, and RGD targeting, these nanoparticles achieve sustained circulation, tumor‑specific accumulation, and robust antitumor efficacy with minimal toxicity. This study supports their further development as a potent therapeutic modality for pancreatic cancer.

Abbreviations

• CCK‑8: Cell Counting Kit‑8
• FBS: Fetal Bovine Serum
• FITC: Fluorescein Isothiocyanate
• HSA: Human Serum Albumin
• PEG: Polyethylene Glycol
• RGD: Arginine–Glycine–Aspartate
• RV: Resveratrol
• PBS: Phosphate‑Buffered Saline