Purpose: Non-viral gene vectors must overcome multiple extracellular and intracellular barriers: stability in blood, efficient cellular uptake, endosomal escape, and intracellular gene release. Stimuli-responsive carriers can address some barriers, but a carrier capable of overcoming multiple barriers simultaneously is still needed. Polydopamine nanoparticles (PDANP) have strong photothermal conversion ability, and pH-responsive boronate esters can detach PEG in acidic endosomes.
Hypothesis: If polydopamine nanoparticles are modified with low-molecular-weight PEI (PEI₁.₈ₖ) and pH-responsive PEG-phenylboronic acid (PEG-PBA) to form PDANP-PEI-rPEG, then the carrier will remain stable at pH 7.4, detach PEG and release DNA at endosomal pH (<6.5), and NIR irradiation will generate local heat to promote endosomal escape, thereby enhancing gene transfection.
Aims: Synthesize pH-responsive PDANP-PEI-rPEG, non-responsive PDANP-PEI-nPEG, and non-PEGylated PDANP-PEI. - Characterize nanoparticle size, zeta potential, DNA condensation, pH-responsive stability, and photothermal conversion. - Evaluate cytotoxicity, cellular uptake, and gene transfection in HepG2 cells with and without NIR irradiation. - Investigate mechanism using Bafilomycin A1 inhibition and confocal microscopy. - Test in vivo photothermal effect in a B16F10 tumor-bearing mouse model.
Delivery system:
Component: Core; Details: Polydopamine nanoparticles (PDANP)
Component: Modifications; Details: PEI₁.₈ₖ; PEG-PBA (pH-responsive boronate ester) or PEG-NH₂ (non-responsive control)
Component: Payload; Details: pGL-3 plasmid DNA; Cy3-labeled DNA for uptake
Component: Complex size; Details: 200–300 nm at mass ratio >0.3
Component: Zeta potential; Details: ~+21 mV at mass ratio 1.5
Component: Photothermal agent; Details: PDANP core; 808 nm NIR laser
Component: Targeting ligand; Details: None
Component: Key feature; Details: pH-triggered PEG detachment plus NIR-triggered endosomal escape
Approach: In vitro: Human hepatoblastoma HepG2 cells; MTT cytotoxicity, flow cytometry uptake, luciferase transfection, Bafilomycin A1 inhibition, CLSM. - In vivo: B16F10 subcutaneous tumor in nude mice; intratumoral injection of PDANP-PEI-rPEG/pGL-3; NIR irradiation; thermal imaging. - Controls: PEI₂₅ₖ, PEI₁.₈ₖ, Lipofectamine 2000, PDANP-PEI-nPEG, PDANP-PEI. - No in vivo gene therapy efficacy, survival, or tumor inhibition study.
Key methods: Physicochemical characterization: DLS, TEM, FT-IR, ¹H NMR. - DNA condensation: Gel retardation assay. - pH-responsive stability: DLS at pH 6.0 vs 7.4 in 10% FBS. - Photothermal conversion: 808 nm laser, thermal imager. - Cytotoxicity: MTT assay. - Cellular uptake: Flow cytometry with Cy3-DNA. - Transfection: Luciferase reporter assay (RLU/mg protein). - Mechanism: Bafilomycin A1 inhibition; CLSM for endosomal escape. - In vivo: Thermal imaging of tumor sites.
Key results: Size / zeta: PDANP 154.4 nm; modified nanoparticles 167–191 nm; complexes 200–300 nm; zeta ~+21 mV at ratio 1.5. - pH responsiveness: At pH 6.0, PDANP-PEI-rPEG/DNA size increased to ~350 nm; non-responsive nPEG unchanged; both stable at pH 7.4. - Photothermal conversion: Temperature increased ~35°C after 20 min NIR at 48 µg/mL; efficiency 54.9% (rPEG) and 52.8% (nPEG). - Cytotoxicity: >80% cell viability for PDANP-PEI-rPEG/DNA and nPEG; PEI₂₅ₖ 66%; Lipofectamine 2000 61%. - Uptake: ~70% Cy3-positive cells; similar to PEI₂₅ₖ. - Transfection: PDANP-PEI-rPEG/DNA was ~100-fold higher than nPEG at same mass ratio; after NIR, doubled and exceeded Lipofectamine 2000. - Bafilomycin A1: Transfection dropped from 4.6×10¹⁰ to 1.2×10⁸ RLU/mg; NIR increased transfection 4.1-fold after Baf A1 vs 2-fold without. - CLSM: NIR separated DNA (red) from endosomes (green), confirming endosomal escape. - In vivo: Tumor site temperature increased 15°C vs 8°C control after 2 min NIR at 0.3 W/cm².
Interpretation: The authors claim PDANP-PEI-rPEG overcomes multiple gene delivery barriers: stability in circulation, pH-responsive PEG detachment in endosomes, and NIR-triggered endosomal escape. It achieves high transfection with low toxicity, outperforming Lipofectamine 2000 under NIR, and is promising for synergistic gene/photothermal cancer therapy.
Limitations: In vivo only photothermal effect: No gene therapy efficacy, tumor inhibition, or survival data. - No biodistribution, long-term toxicity, or immune response data. - No targeting ligand: Delivery relies on local injection and passive accumulation. - Limited cell lines: HepG2 in vitro; B16F10 only for in vivo thermal study. - Therapeutic gene not tested: p53 studies are ongoing; pGL-3 reporter only. - NIR penetration and safety not fully evaluated. - Citation details incomplete in the supplied excerpt.
