International Journal of Pharmaceutics (inferred; the supplied PDF is marked “2019ResearchNon-viral Gene Delivery
Hyaluronic acid-coated pH sensitive poly (β-amino Ester) nanoparticles for codelivery of embelin and TRAIL plasmid for triple negative breast cancer treatment
Yingqi Xu, Dingxin Liu, Jie Hu, Peirong Ding, Meiwan Chen
Summary
Triple-negative breast cancer (TNBC) lacks effective targeted therapy. Nonviral co-delivery of an anticancer drug and a therapeutic gene may improve treatment, but efficient tumor-targeted co-delivery remains challenging. TRAIL has shown limited clinical benefit alone, and embelin (EMB) can inhibit XIAP to promote apoptosis. Nanoparticle properties: PPNP CMC = 0.0065 mg/mL. HA coating reduced hemolysis from 37.72% to <0.5%. HA/PPNP remained stable in 10% FBS for 24 h, while PPNP aggregated to ~4000 nm. - Uptake: HA/PPNP increased Nile red.
Keywords
Purpose: Triple-negative breast cancer (TNBC) lacks effective targeted therapy. Nonviral co-delivery of an anticancer drug and a therapeutic gene may improve treatment, but efficient tumor-targeted co-delivery remains challenging. TRAIL has shown limited clinical benefit alone, and embelin (EMB) can inhibit XIAP to promote apoptosis.
Hypothesis: Hyaluronic acid (HA)-coated, pH-sensitive poly(β-amino ester)–polyethyleneimine (PBAE-PEI) nanoparticles can co-deliver embelin and TRAIL plasmid (pTRAIL). HA will target CD44-overexpressing TNBC cells, the pH-sensitive PBAE core will release cargo in acidic tumor environments, and combined EMB/pTRAIL will synergistically induce cytotoxicity and apoptosis.
Aims: Synthesize and characterize PBAE-PEI copolymer and HA-coated nanoparticles. - Co-load EMB and pTRAIL and evaluate pH sensitivity, stability, hemolysis, and pTRAIL condensation. - Assess CD44-mediated cellular uptake in MDA-MB-231 TNBC vs. MCF-7 non-TNBC cells. - Evaluate cytotoxicity, apoptosis, caspase 3/7 activity, ROS generation, and apoptosis-related protein expression.
Delivery system: Polymer: Amphiphilic PBAE-PEI copolymer. PBAE synthesized from 1,6-hexanediol diacrylate and 5-amino-1-pentanol (1.2:1 molar ratio) via Michael addition, then conjugated with branched PEI (1.8 kDa). - Nanoparticle type: Solvent-evaporation nanoparticles; hydrophobic EMB loaded into the PBAE core; pTRAIL complexed with cationic PEI; HA coated electrostatically. - Targeting ligand: Hyaluronic acid (HA) for CD44. - Payload: Embelin (EMB) and pTRAIL plasmid (pEGFP-TRAIL). - Formulation: HA:PPNP ratio 1:2 w/w selected. PPNP size = 84.69 ± 7.6 nm, zeta = +34.3 ± 3.89 mV; HA/PPNP size = 182.7 ± 2.7 nm, zeta = −11.5 ± 1.32 mV. EMB EE = 57.92 ± 1.14%, DL = 7.24 ± 0.93%. Complete pTRAIL retardation at mass ratio 20/1. - pH sensitivity: PBAE is hydrophobic at neutral pH and hydrophilic under acidic conditions; Nile red release increased as pH decreased.
Approach: In vitro only. Cell lines: MDA-MB-231 (TNBC, high CD44) and MCF-7 (non-TNBC, lower CD44). - Treatments: free EMB, pTRAIL-HA/PPNP, EMB-PPNP, EMB-HA/PPNP, EMB/pC3-HA/PPNP (control plasmid), and EMB/pTRAIL-HA/PPNP. - Doses: equivalent to 1 µg/mL pTRAIL or pC3, 10 µM EMB, or combination. IC50 of EMB-HA/PPNP at 48 h = 10.24 ± 1.44 µM. - n ≥ 3 independent experiments; one-way ANOVA or t-test; p < 0.05 significant. - No in vivo model.
Key methods: ¹H NMR and GPC for polymer structure/molecular weight. - DLS and TEM for nanoparticle size, zeta potential, and morphology. - HPLC for EMB encapsulation efficiency and drug loading. - Agarose gel retardation for pTRAIL condensation. - Pyrene fluorescence for critical micelle concentration (CMC). - Nile red release for pH sensitivity. - Hemolysis assay and 10% FBS stability for biocompatibility/stability. - Flow cytometry and Incell Analyzer for cellular uptake. - MTT assay for cytotoxicity. - Annexin V/PI for apoptosis. - Caspase-Glo 3/7 assay for caspase activity. - DCFH2-DA for ROS. - Western blot for XIAP, cleaved caspase 3, cleaved PARP, and GAPDH.
Key results: Nanoparticle properties: PPNP CMC = 0.0065 mg/mL. HA coating reduced hemolysis from 37.72% to <0.5%. HA/PPNP remained stable in 10% FBS for 24 h, while PPNP aggregated to ~4000 nm. - Uptake: HA/PPNP increased Nile red and YOYO-1 uptake in both cell lines vs. free payload. HA pretreatment significantly diminished uptake in MDA-MB-231 cells but not MCF-7 cells. - Cytotoxicity: In MDA-MB-231 cells, EMB/pTRAIL-HA/PPNP induced 75.38% cytotoxicity vs. 31.68% for EMB-HA/PPNP and 19.95% for pTRAIL-HA/PPNP. - Apoptosis: Apoptotic rates: control 7.71%, pTRAIL-HA/PPNP 16.85%, free EMB 31.26%, EMB-PPNP 41.04%, EMB-HA/PPNP 53.52%, EMB/pC3-HA/PPNP 51.15%, EMB/pTRAIL-HA/PPNP 70.19%. - ROS: EMB/pTRAIL-HA/PPNP increased ROS to 2.76-fold vs. 1.28-fold for pTRAIL-HA/PPNP and 1.41-fold for free EMB. - Protein expression: EMB/pTRAIL-HA/PPNP decreased XIAP and increased cleaved caspase 3 and cleaved PARP.
Interpretation: HA/PPNP is a potential vector for co-delivery of anticancer drugs and therapeutic genes. The combination of EMB and pTRAIL delivered by HA/PPNP synergistically enhances cytotoxicity and apoptosis in TNBC cells and may be a promising strategy for TNBC therapy.
Limitations: In vitro only; no in vivo efficacy, biodistribution, or toxicity data. - No normal breast epithelial cell control; MCF-7 is non-TNBC but not normal. - Long-term storage stability and large-scale manufacturing were not evaluated. - HA targeting is inferred from CD44 expression and competitive inhibition; no direct CD44 blocking or receptor knockdown. - Mechanism studies focused mainly on MDA-MB-231 cells. - No pharmacokinetic or systemic toxicity assessment.
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