Polymeric nanoparticles as cancer-specific DNA delivery vectors to human hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the third most deadly cancer in the US, with a meager 5-year survival rate of <20%. Such unfavorable numbers are closely related to the heterogeneity of the disease and the unsatisfactory therapies currently used to manage patients with invasive HCC. Outside of the clinic, gene therapy research is evolving to overcome the poor responses and toxicity associated with standard treatments. The inadequacy of gene delivery vectors, including poor intracellular delivery and cell specificity, are major barriers in the gene therapy field. Herein, we described a non-viral strategy for effective and cancer-specific DNA delivery to human HCC using biodegradable poly(beta-amino ester) (PBAE) nanoparticles (NPs). Varied PBAE NP formulations were evaluated for transfection efficacy and cytotoxicity to a range of human HCC cells as well as healthy human hepatocytes. To address HCC heterogeneity, nine different sources of human HCC cells were utilized. The polymeric NPs composed of 2-((3-aminopropyl)amino) ethanol end-modified poly(1,5-pentanediol diacrylate-co-3-amino-1-propanol) ('536') at a 25 polymer-to-DNA weight-to-weight ratio led to high transfection efficacy to all of the liver cancer lines, but not to hepatocytes. Each individual HCC line had a significantly higher percentage of exogenous gene expression than the healthy liver cells (P<0.01). Notably, this biodegradable end-modified PBAE gene delivery vector was not cytotoxic and maintained the viability of hepatocytes above 80%. In a HCC/hepatocyte co-culture model, in which cancerous and healthy cells share the same micro-environment, 536 25 w/w NPs specifically transfected cancer cells. PBAE NP administration to a subcutaneous HCC mouse model, established with one of the human lines tested in vitro, confirmed effective DNA transfection in vivo. PBAE-based NPs enabled high and preferential DNA delivery to HCC cells, sparing healthy hepatocytes. These biodegradable and liver cancer-selective NPs are a promising technology to deliver therapeutic genes to liver cancer.

Keywords: Carcinoma, hepatocellular; Gene therapy; Nanoparticle.

Fig. 1

A. Synthesis of end-capped PBAE polymers. A diacrylate backbone monomer (B), an amino-alcohol sidechain monomer (S) and an amine containing end-capping molecule (E) were conjugated through a two-step process. B. Chemical structures of B, S and E monomers used in the synthesis of PBAE polymers for this study.

Fig. 2

Viability of hepatocytes (THLE-3) 24 hours after transfection with a broad range of PBAE structures and concentrations. PBAE polymers were evaluated in comparison to three commercially available non-viral transfection vectors (positive controls) at a range of dosages. Viability of cells treated with polymer 536 at a 25 polymer-to-DNA w/w ratio remained above 80%. Among the positive controls, only jetPRIME^™ and PEI 25kDa at relatively low concentrations enabled comparably high viability. Results from each condition were obtained as the average metabolic activity relative to untreated controls. The proportions of Lipofectamine^™ 2000-to-DNA and jetPRIME^™-to-DNA in each condition are expressed as weight-to-weight and weight-to-volume ratios, respectively.

Fig. 3

Transfection efficacy screen of all PBAE and positive control NPs to A. Two representatives of HCC cell lines and the THLE-3 hepatocytes. EGFP expression was measured using flow cytometry 48 hours after transfection and analyzed for the percentage (positive %) and intensity (geometric mean) of GFP expression. Geometric mean results from each condition are relative to untreated controls. Norm.: Normalized. * P < 0.05 for statistically significant differences between PBAE polymers with superior transfection efficacy and the most effective among positive controls for each cell line.

Fig. 4

Transfection efficacy of the optimal PBAE (536 25 w/w) and positive control (PEI 25kDa 2 w/w) NP formulations for all cell lines. Transfection of eGFP DNA with 536 25 w/w NPs was effective and consistent to all cancer cells. In addition, the eGFP expression in all HCC lines was also specific over healthy hepatocytes. While also cancer-selective, PEI 25kDa 2 w/w NPs did not promote consistently high eGFP expression to all HCC cells. A. EGFP expression of all cell lines 48 hours after transfection with 536 25 w/w (left) or PEI 25kDa 2 w/w (right). B. Flow cytometry gating and microscopy images of two representatives of HCC lines and THLE-3 hepatocytes treated with 536 25 w/w (left column) or PEI 25kDa 2 w/w (right column). * P < 0.01

Fig. 5

A. Levels of eGFP expression following electroporation for HCC cells and hepatocytes. B. Cy5 signal from all nine cancer cell populations and THLE-3 hepatocytes showing cellular uptake of 536 NPs. * P < 0.05 for statistically significant differences between hepatocytes and each individual HCC line.

Fig. 6

Both efficacy and cancer-specificity of 536 25 w/w NPs were preserved in co-culture of HCC (Huh-7) and hepatocytes (THLE-3). A. Microscopy images and B. Flow cytometry gating of a 536 25 w/w NP-treated co-culture representative 48 hours after eGFP transfection. Microscopy images show: merged eGFP and RFP channels (top left), eGFP channel only (top right), bright-field only (bottom left), and RFP channel only (bottom right). C. Percentage of GFP positive cells among Huh-7 (RFP positive) and THLE-3 (RFP negative) by flow cytometry analysis. Co-cultures were plated and transfected in triplicate. * P < 0.05

Fig. 7

A. Chemical structure of the polymer 2-((3-aminopropyl)amino)ethanol end-modified poly(1,5-pentanediol diacrylate-co-3-amino-1-propanol) (536). B. TEM image and C. Physical characterization (size and zeta-potential) of 536-based NPs carrying eGFP plasmid DNA at the 25 w/w ratio. Imaging, sizing and zeta-potential measurement were carried out with NPs prepared following the same methods described for transfection experiments and immediately after NP complexation. A 1:1 v/v dilution of NP mix to PBS 1x was performed for sizing and zeta-potential measurements.

Fig. 8

Effective DNA delivery in vivo using an optimal PBAE formulation. A. Bioluminescence images of subcutaneous Huh-7 xenograft mice at 6, 24 and 48 hours following intratumoral injection of 536 25 w/w NPs or PBS. B. Summary analysis of the average radiance for treated and control animals at the three recorded time points. * P < 0.05