Purpose: Branching architecture is a tunable parameter for polymeric gene carriers, but how the degree of branching (DB) of bioreducible poly(amido amine)s (PAAs) affects pDNA binding, cellular uptake, endocytosis pathways, and transfection efficiency was not well defined.
Hypothesis: If PAAs share the same repeating units and molecular weight but differ in branching degree, then DB will strongly affect pDNA compaction, cellular uptake, and gene transfection efficiency. An intermediate/low-branched architecture may outperform both linear and highly branched analogues.
Aims: Synthesize linear (LPAA), low-branched (LBPAA), and high-branched (HBPAA) bioreducible PAAs with similar molecular weights but different DBs. - Characterize pDNA condensation, particle size, zeta potential, and bioreducible release. - Evaluate cytotoxicity, cellular uptake, endocytosis pathways, and in vitro transfection efficiency in OVCAR-3 cells. - Identify the optimal branching degree for non-viral gene delivery.
Delivery system:
Component: Polymer class; Details: Bioreducible poly(amido amine)s (PAAs) containing disulfide linkages
Component: Variants; Details: LPAA (linear, DB = 0), LBPAA (low-branched, DB = 0.10), HBPAA (high-branched, DB = 0.42)
Component: Molecular weight; Details: ~4 kDa (MALDI-TOF: LPAA 4,758; LBPAA 4,154; HBPAA 4,358)
Component: Payload; Details: pGL4 luciferase reporter plasmid DNA
Component: Complexation; Details: Electrostatic self-assembly; polymer/DNA weight ratios varied (1–200)
Component: Bioreducible feature; Details: Disulfide bonds cleaved by DTT (10 mM), releasing pDNA
Component: Targeting ligand; Details: None
Component: Controls; Details: Linear polyethyleneimine (LPEI, 25 kDa)
Approach: In vitro only. Human ovarian carcinoma OVCAR-3 cells. - Cytotoxicity: 96-well plates, 5,000 cells/well, polymer concentrations 0.0125–1 mg/mL, 4 h exposure + 44 h recovery, MTT assay. - Transfection: 20,000 cells/well, pDNA dose 10 µg/mL, 4 h in FBS-free medium, then 44 h in complete medium; luciferase assay. - Uptake/mechanism: Confocal microscopy with Cy5-labeled pDNA and acridine orange nuclear stain; flow cytometry; endocytosis inhibitors chlorpromazine (clathrin), Filipin III (caveolae), wortmannin (PI3K/macropinocytosis). - No in vivo model. No targeting ligand. No tumor regression or survival data.
Key methods: Gel electrophoresis: pDNA condensation and DTT-triggered release. - Dynamic light scattering (DLS): particle size. - Zeta potential: surface charge. - Ethidium bromide (EB) exclusion assay: pDNA binding affinity. - MTT assay: cytotoxicity. - Luciferase assay: transfection efficiency. - Confocal laser scanning microscopy: intracellular pDNA localization. - Flow cytometry: quantification of cellular uptake. - Endocytosis inhibitors: pathway identification.
Key results: pDNA condensation: HBPAA completely retarded pDNA at polymer/DNA ratio 1; LBPAA at 12.5; LPAA at 25. DTT (10 mM) released free pDNA, confirming bioreducible behavior. - Particle size: Only LBPAA condensed pDNA into ~130 nm nanoparticles at ratio 12.5. HBPAA formed ~700 nm polyplexes across ratios 12.5–100. LPAA formed large/increasing particles, then ~400 nm at ratio 100. - Zeta potential: LBPAA and HBPAA became positive at ratios >5; LPAA required ratio >20. LPAA zeta values were ~7 mV lower than branched PAAs at same ratios. - Cytotoxicity: PAAs showed ≥80% OVCAR-3 survival up to 1 mg/mL; LPEI 25k caused ~90% cell death at 0.25 mg/mL. - Transfection: LBPAA achieved the highest efficiency, peaking at polymer/DNA ratio 100, comparable to LPEI 25k. HBPAA was lower at low ratios but comparable at high ratios. LPAA was poor. - Cellular uptake: At ratio 25, LBPAA > HBPAA; at ratio 100, LBPAA ≈ HBPAA, and both exceeded LPEI 25k. LPAA uptake was negligible. - Endocytosis pathway: Chlorpromazine had no effect; Filipin III and wortmannin strongly reduced transfection; combination reduced to <20%, indicating caveolae- and PI3K/macropinocytosis-mediated uptake, not clathrin-mediated.
Interpretation: The authors claim that branching degree of bioreducible PAAs strongly influences pDNA binding, cellular uptake, and transfection. Low-branched PAA (LBPAA) showed the best combination of pDNA binding, nanoparticle size, cellular uptake, and transfection efficiency, outperforming linear and highly branched analogues. This provides a strategy to tune polymer architecture for non-viral gene delivery.
Limitations: In vitro only: No in vivo validation, biodistribution, or therapeutic efficacy. - Single cell line: OVCAR-3 ovarian cancer cells only. - No targeting ligand: Delivery relies on nonspecific electrostatic interactions. - Short-term assays: Transfection measured at 48 h; long-term expression and safety not assessed. - FBS-free transfection: 4 h in serum-free medium; may not fully reflect in vivo serum conditions. - Low molecular weight: PAAs ~4 kDa; suitability for systemic delivery and stability in circulation not established. - No direct comparison of endocytosis rate: Inhibitor studies identify pathways but do not quantify uptake kinetics. - Citation details incomplete in supplied file (authors/DOI missing).
