Biomaterial-mediated cancer-specific DNA delivery to liver cell cultures using synthetic poly(beta-amino esters)

Purpose: Liver cancer has limited curative options, and transarterial chemoembolization (TACE) provides local access for gene therapy. However, prior nonviral delivery strategies suffered from low in vitro efficacy and high toxicity to healthy liver cells. There is a need for a delivery platform that efficiently transfects hepatoma cells while sparing non-cancer hepatocytes.

Hypothesis: Poly(beta-amino ester) (PBAE)–DNA nanoparticles can deliver plasmid DNA to hepatoma cells with high efficiency and low cytotoxicity, and they will exhibit intrinsic biomaterial-mediated specificity for hepatoma cells over healthy hepatocytes, including in co-culture.

Aims: Synthesize and screen a library of PBAEs to identify lead formulations for DNA delivery to MCA-RH7777 hepatoma cells. - Quantify transfection efficiency, transgene expression, and cytotoxicity in hepatoma vs. hepatocyte monocultures and compare with commercial reagents. - Test whether lead PBAEs show cancer-specific DNA delivery in hepatoma–hepatocyte co-culture using eGFP and luciferase reporters. - Identify formulations with low toxicity to healthy hepatocytes for potential liver cancer gene therapy.

Delivery system: Polymer: Synthetic poly(beta-amino esters) (PBAEs) made from backbone diacrylates (B3–B6), amino-alcohol side chains (S3–S6), and amine end-caps (E3, E6, E7). Lead examples include 457e, 1.1:1 (B4-S5-E7, ether-purified); 537e, 1.05:1; 447e; 453e; 456e. - Nanoparticle: PBAE–DNA polyplexes formed in 25 mM sodium acetate buffer (pH 5) at varying polymer:DNA w/w ratios (25, 50, 75, 90). - Payload: Plasmid DNA encoding eGFP, luciferase, H2B-Cherry, or Piggybac transposase. - Targeting: No targeting ligand; specificity is attributed to intrinsic PBAE/cell-type interactions.

Approach: In vitro monoculture: Buffalo rat hepatoma MCA-RH7777 and hepatocyte BRL-3A cells; 96-well format, 600 ng DNA/well, 4 h incubation. Controls: Lipofectamine 2000, X-tremeGENE DNA HP, and PEI. - Co-culture: H2B-Cherry-labeled MCA-RH7777 cells mixed 1:1 with BRL-3A cells, transfected, and analyzed by flow cytometry. - No in vivo model. Viability assessed 24 h post-transfection; expression assessed 48 h post-transfection.

Key methods: Flow cytometry: % eGFP+ cells and geometric mean fluorescence intensity (PI exclusion for dead cells). - Luciferase BrightGlo assay for expression. - MTS assay and DAPI-based automated cell counting for viability/cytotoxicity. - Fluorescence microscopy for transgene expression and co-culture distribution. - FACS sorting and FlowJo gating for labeled hepatoma cells.

Key results: Hepatoma transfection: 457e, 1.1:1 achieved up to 98 ± 0.4% eGFP transfection in MCA-RH7777 cells with no measurable cytotoxicity. - Hepatocyte transfection/toxicity: BRL-3A transfection reached 73 ± 0.4% with 10 ± 4% nonspecific cytotoxicity; commercial reagents caused 30–90% toxicity in BRL-3A at doses needed for >50% transfection. - Specificity in monoculture: 12 of 21 PBAE formulations showed significant hepatoma-over-hepatocyte specificity (P < 0.05 for both % transfected and eGFP intensity). - Luciferase specificity: Top polymers gave 220 ± 30-fold and 470 ± 30-fold higher hepatoma specificity; best condition (537e, 1.05:1, 75 w/w) showed 470 ± 25-fold more luminescence in hepatoma cells. - Co-culture specificity: 1.9 ± 0.1- to 5.8 ± 1.4-fold more transfected hepatoma than hepatocytes by percentage; up to 530 ± 60-fold higher average eGFP expression per cell in hepatoma cells. One top formulation gave 95 ± 0.2% hepatoma transfection vs 27 ± 0.2% hepatocyte transfection with 96 ± 9% relative hepatocyte viability.

Interpretation: PBAE nanoparticles are a viable nonviral strategy for liver cancer gene therapy, delivering DNA to nearly 100% of hepatoma cells while maintaining high intrinsic specificity and low toxicity to healthy hepatocytes. This supports further in vivo/clinical translation for hepatocellular carcinoma and potentially other hepatic diseases.

Limitations: Entirely in vitro; no animal or clinical validation. - Mechanism of cancer-cell specificity is not fully understood. - Co-culture is 2D and may not fully mimic the in vivo tumor microenvironment. - Uses a CMV promoter with no transcriptional targeting; specificity relies on delivery. - Long-term safety, immunogenicity, and clearance were not evaluated. - Cell lines are rat-derived, not human hepatocellular carcinoma.