Polymeric nanoparticles for dual-targeted theranostic gene delivery to hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) develops predominantly in the inflammatory environment of a cirrhotic liver caused by hepatitis, toxin exposure, or chronic liver disease. A targeted therapeutic approach is required to enable cancer killing without causing toxicity and liver failure. Poly(beta-amino-ester) (PBAE) nanoparticles (NPs) were used to deliver a completely CpG-free plasmid harboring mutant herpes simplex virus type 1 sr39 thymidine kinase (sr39) DNA to human HCC cells. Transfection with sr39 enables cancer cell killing with the prodrug ganciclovir and accumulation of 9-(4-¹⁸F-fluoro-3-hydroxymethylbutyl)guanine (¹⁸F-FHBG) for in vivo imaging. Targeting was achieved using a CpG-free human alpha fetoprotein (AFP) promoter (CpGf-AFP-sr39). Expression was restricted to AFP-producing HCC cells, enabling selective transfection of orthotopic HCC xenografts. CpGf-AFP-sr39 NP treatment resulted in 62% reduced tumor size, and therapeutic gene expression was detectable by positron emission tomography (PET). This systemic nanomedicine achieved tumor-specific delivery, therapy, and imaging, representing a promising platform for targeted treatment of HCC.

Fig. 1.. Schema describing a tumor-targeted theranostic approach for the treatment of HCC.

PBAE 536 NPs are synthesized with a CpG-free transcriptionally targeted plasmid encoding for sr39. DNA delivery to healthy cells does not result in therapeutic gene expression.

Fig. 2.. PBAE 536 NPs transfect liver cancer cells with a reporter gene in vitro.

(A) Structure of polymer PBAE 536. (B) TEM image of PBAE 536 NPs. Scale bars, 100 nm. (C) In vitro transfection efficacy of Hep3b cells as quantified by flow cytometry. (D) Viability of Hep3b cells transfected with varying doses of GFP DNA. PBAE NPs were synthesized with PBAE 536 at a polymer:DNA mass ratio of 25 (w/w). Means ± SE of the mean are shown (n = 3). Statistically significant transfection was calculated by one-way ANOVA with Dunnett’s multiple comparisons test compared to untreated cells. (E) Fluorescence micrographs of GFP expression in transfected Hep3b cells. Scale bars, 500 μm. ***P < 0.001; ****P < 0.0001.

Fig. 3.. CpG-free sr39 plasmids induce sr39tk expression in vitro without elevated TLR9 activation.

(A and B) Plasmid maps are shown for CMV-sr39 and AFP-sr39. (C) hTLR-9 activation in HEK-Blue reporter cells after exposure to CpG-containing and CpG-free CMV-sr39 and (D) AFP-sr39 plasmid DNA. Differences in TLR9 activation between unmodified and CpG-free plasmids were determined by two-way ANOVA and Sidak’s multiple comparisons test. (E) qRT-PCR analysis of sr39 expression in transfected cells. (F) Hep3b cellular viability 5 days after transfection with sr39 NPs and treatment with GCV (1.25 μg/ml). Differences among DNAs were determined by two-way ANOVA and Sidak’s multiple comparisons test. Means ± SE are shown for all graphs (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 4.. AFP transcriptional targeting restricts sr39-mediated cell death to AFP-producing HCC cells.

(A) AFP expression in fixed and permeabilized Huh7, Hep3b, SK-HEP-1, PC-3, and THLE-3 cells measured by flow cytometry. (B and C) Transfection efficacy and viability of cell lines transfected with GFP DNA (600 ng per well) using PBAE 536 NPs. Statistically significant differences between cell lines were determined using one-way ANOVA with Tukey’s multiple comparisons test. (D to H) Viability time course of cells transfected with CpG-free CMV-sr39 and **AFP-sr39 DNA and treated with GCV (1.25 mg/ml). Loss in viability for each DNA was calculated by two-way ANOVA with Dunnett’s multiple comparisons between each time point and day 1. Means ± SE are shown for all graphs (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. n.s., not significant.

Fig. 5.. sr39 NPs enable specific accumulation of ¹⁸F-FHBG in target HCC cells.

(A to E) Cellular radioactivity in transfected cells incubated with 10 μCi of ¹⁸F-FHBG for 1 hour, normalized to total protein content. Differences were calculated by one-way ANOVA with Dunnett’s multiple comparisons test between treatment groups and controls. (F and G) Fold radioactivity accumulation in cancer cells normalized to THLE-3 cells treated with the same NP. Means ± SE are shown for all graphs (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. n.s., not significant.

Fig. 6.. Intravenously administered NPs efficiently transfect orthotopic HCC tumors.

(A) NP biodistribution shown as average radiance (p/s/cm²/sr) of major organs 1 hour after intravenous fluorescent NP administration. Significant differences between organ radiance were calculated by Kruskal-Wallis nonparametric test and Dunn’s test for multiple comparisons. *P < 0.05. Data are shown as means ± SE (n = 3). (B) Representative image of NP biodistribution 1 hour after intravenous fluorescent NP administration. (C) Distribution of transgene expression after intravenous administration of fLuc NPs, shown as average radiance. Differences in radiance between liver and tumor tissue were determined by a ratio-paired t test between average radiance over the region of interest. *P < 0.05. Data shown as means ± SE (n = 3). (D) Representative images of orthotopic tumor and liver transfected with fLuc NPs administered by intravenous injection. (E) Fold sr39 expression in organs after intravenous administration of CMV-sr39 NPs or (F) AFP-sr39 NPs by qRT-PCR. Significant differences between tumor and healthy tissue were calculated by one-way ANOVA and Dunnett’s test for multiple comparisons. *P < 0.05 and **P < 0.01, corrected for multiple comparisons. Data are shown as means ± SE (n = 3).

Fig. 7.. CpGf-sr39 NP treatment significantly inhibits tumor growth and enables monitoring by PET/CT.

Mice with orthotopic xenograft Hep3b tumors were treated with intravenous administration of fLuc NPs (n = 11), CpGf-CMV-sr39 NPs (n = 10), or CpGf-AFP-sr39 NPs (n = 8) with systemic GCV. (A) After 16 days, tumors treated with CpGf-AFP-sr39 had significantly smaller tumors than the other two groups. Differences between treatment groups were determined by one-way ANOVA with Tukey’s posttests among the three groups. *P < 0.05. (B) Representative images show differences in tumor size within the liver. (C) Representative PET/MRI imaging shows intratumoral ¹⁸F-FHBG activity in a tumor-bearing mouse treated with CpGf-AFP-sr39. Scale bar, 1 cm. (D) Activity in major organs with fLuc (n = 2), CpGf-CMV-sr39 (n = 2), or CpGf-AFP-sr39 (n = 3) NPs and subsequent ¹⁸F-FHBG injection. Activity was calculated per mass of tissue and then normalized to activity in the control group (fLuc NP). Significant differences between treatment groups were determined by two-way ANOVA and Tukey’s posttest. ***P < 0.001. All data shown as means ± SE.