Stepwise pH-responsive nanoparticles containing charge-reversible pullulan-based shells and poly(β-amino ester)poly(lactic-co-glycolic acid) cores as carriers of anticancer drugs for combination therapy on he

Purpose: Hepatocellular carcinoma (HCC) has poor prognosis and limited effective systemic therapy. Combination antiangiogenesis and chemotherapy with paclitaxel (PTX) and combretastatin A4 (CA4) is synergistic, but both drugs have poor solubility, lack tumor targeting, and cause systemic toxicity. A tumor microenvironment–responsive carrier is needed to co-deliver them selectively to HCC.

Hypothesis: A stepwise pH-responsive nanoparticle with a charge-reversible pullulan-based shell and a poly(β-amino ester)/poly(lactic-co-glycolic acid) (PBAE/PLGA) core will target HCC via asialoglycoprotein receptor (ASGPR) and the enhanced permeability and retention (EPR) effect. The shell will detach at weakly acidic tumor pH (~6.5), exposing the cationic core for cell uptake; the core will then release PTX and CA4 at endo/lysosomal pH (~5.5), enhancing synergistic antiangiogenic/chemotherapeutic efficacy while reducing toxicity.

Aims: Synthesize and characterize charge-reversible pullulan (CAPL) and PBAE/PLGA nanoparticles. - Prepare CAPL/PBAE/PLGA nanoparticles loaded with PTX and CA4 and evaluate pH-responsive charge reversal and drug release. - Assess cellular uptake, intracellular trafficking, cytotoxicity, and anti-migration effects in HepG2 HCC cells and HUVECs under different pH conditions. - Evaluate in vivo biodistribution, tumor targeting, antitumor efficacy, and antiangiogenic activity in HepG2 tumor-bearing mice.

Delivery system: Core: PBAE + PLGA at 5/1 w/w. PBAE synthesized from 4,4′-trimethylenedipiperidine and 1,4-butanediol diacrylate (Mw ~28,000); PLGA Mw ~20 kDa, 50/50 lactide/glycolide. - Shell: CAPL, a charge-reversible pullulan derivative. Pullulan was modified with diethylenetriamine to AMPL, then carboxylated with cis-4-cyclohexene-1,2-dicarboxylic anhydride. β-Carboxylic amide bonds cleave under weakly acidic conditions, reversing charge from negative to positive and detaching the shell. - Payload: PTX and CA4; Dil and Cy5.5 used for imaging. - Formulation: Drug/PBAE/PLGA feed ratio 9/25/5 (w/w/w); CAPL coating at CAPL/PBAE/PLGA 15/5/1. Final size ~178.1 nm, zeta potential −17.8 mV. PTX and CA4 loading contents were 8.2% and 6.9%, respectively. - Targeting ligand: Pullulan shell targets ASGPR overexpressed on HCC cells; additional passive tumor accumulation via EPR.

Approach: In vitro: HepG2 HCC cells and human umbilical vein endothelial cells (HUVECs) cultured at pH 7.4, 7.0, and 6.5. Treatments included free PTX, free CA4, PTX/CA4 mixture, and PTX/CA4 nanoparticles. MTT cytotoxicity, wound healing migration, flow cytometry, and confocal microscopy were used. - In vivo: BALB/c nude mice bearing subcutaneous HepG2 xenografts. Biodistribution was assessed with Cy5.5-labeled nanoparticles by IVIS at 6 and 24 h, and with Dil by cryosection confocal microscopy at 24 h. Antitumor study: 5 groups (n = 6/group) treated with saline, free PTX, free CA4, PTX/CA4 mixture, or PTX/CA4 nanoparticles via tail vein every other day for 4 injections at PTX 8 mg/kg and CA4 10 mg/kg. Tumor volume and body weight measured every 2 days; tumors/organs harvested for H&E and CD31 immunohistochemistry. - Additional model: MCF-7 tumor-bearing mice were used to assess distribution in a non-ASGPR-expressing tumor.

Key methods: FT-IR, ¹H NMR, and pH-metric titration for CAPL characterization. - DLS and TEM for nanoparticle size, PDI, zeta potential, and morphology. - Zeta potential and TEM after incubation at pH 7.4, 7.0, and 6.5 to assess charge reversal and shell detachment. - UV spectrophotometry and UPLC for drug loading and in vitro release at pH 7.4, 7.0, 6.5, and 5.5. - Flow cytometry and confocal microscopy for cellular uptake and intracellular localization; LysoTracker for endo/lysosomal escape. - MTT assay for cytotoxicity; wound healing assay for HUVEC migration. - IVIS imaging for in vivo biodistribution; confocal cryosection for tissue accumulation. - H&E staining and CD31 immunohistochemistry for tissue toxicity and microvessel density.

Key results: Nanoparticle properties: PBAE/PLGA nanoparticles were ~120 nm; CAPL/PBAE/PLGA nanoparticles were ~178.1 nm with zeta potential −17.8 mV and a core–shell structure with 20–50 nm shell. - pH-responsive charge reversal: At pH 6.5, zeta potential changed from −17.3 mV to +9.8 mV within 2 h; CAPL shells almost completely detached by 6 h. - Drug release: At pH 5.5, approximately 21.6% of PTX and 70.0% of CA4 were released at 24 h. CA4 released faster than PTX, and release increased as pH decreased. - In vitro efficacy: PTX/CA4 nanoparticles showed higher cytotoxicity than free drugs or mixture in HepG2 cells. In HUVECs, PTX/CA4 nanoparticles showed pH-dependent inhibition, with increased cytotoxicity at pH 6.5. Nanoparticles also inhibited HUVEC migration more effectively than free drugs. - In vivo targeting: CAPL/PBAE/PLGA/Cy5.5 accumulated in tumors at 6 h and was mostly in tumor and liver at 24 h, with reduced liver/spleen distribution compared with PBAE/PLGA/Cy5.5. Tumor accumulation was also observed in MCF-7 tumors. - In vivo antitumor efficacy: PTX/CA4 nanoparticles significantly delayed tumor growth compared with PTX/CA4 mixture. Free PTX and mixture caused body weight loss and hepatic injury; nanoparticle treatment avoided these effects. CD31 staining showed the greatest reduction in microvessel density with PTX/CA4 nanoparticles.

Interpretation: CAPL/PBAE/PLGA nanoparticles provide stepwise pH-responsive, HCC-targeted co-delivery of PTX and CA4. They enhance synergistic antiangiogenesis and chemotherapy, improve tumor accumulation, and reduce systemic toxicity. The system is proposed as a promising novel carrier for antitumor drug delivery in HCC.

Limitations: Subcutaneous HepG2 xenograft model, not orthotopic HCC; no survival study and short follow-up. - PTX and CA4 were loaded into separate nanoparticle preparations and then combined; true single-particle co-encapsulation was not demonstrated. - Small in vivo group size (n = 6); limited quantitative pharmacokinetics and long-term biodistribution. - No long-term toxicity, immunogenicity, or manufacturing/stability assessment. - Tumor targeting may be partly due to EPR and improved stability, since accumulation was also seen in non-ASGPR-expressing MCF-7 tumors. - Clinical translation and human safety remain untested.