Poly(β-amino ester) nanoparticle delivery of TP53 has activity against small cell lung cancer in vitro and in vivo

Abstract

Small cell lung cancer (SCLC) is an aggressive disease with one of the highest case-fatality rates among cancer. The recommended therapy for SCLCs has not changed significantly over the past 30 years; new therapeutic approaches are a critical need. TP53 is mutated in the majority of SCLC cases and its loss is required in transgenic mouse models of the disease. We synthesized an array of biodegradable poly(β-amino ester) (PBAE) polymers that self-assemble with DNA and assayed for transfection efficiency in the p53-mutant H446 SCLC cell line using high-throughput methodologies. Two of the top candidates were selected for further characterization and TP53 delivery in vitro and in vivo. Nanoparticle delivery of TP53 resulted in expression of exogenous p53, induction of p21, induction of apoptosis, and accumulation of cells in sub-G1 consistent with functional p53 activity. Intratumoral injection of subcutaneous H446 xenografts with polymers carrying TP53 caused marked tumor growth inhibition. This is the first demonstration of TP53 gene therapy in SCLC using nonviral polymeric nanoparticles. This technology may have general applicability as a novel anticancer strategy based on restoration of tumor suppressor gene function.

Figure 1. Poly(beta-amino ester) synthesis scheme depicting the conjugate addition of amines to diacrylates in two steps

The three R groups allow modifications to the polymer backbone (R), side chain (R′) and end-groups (R″). Each monomer comprises a backbone (B), a side chain (S) or an end-group (E).

Figure 2. Nanoparticle characterization

The ability of the PBAEs to form nanoparticles was examined using NTA to determine the nanoparticle size (A) and by gel electrophoresis (B). Gel electrophoresis of the nanoparticles shows that at the proper PBAE:DNA weight:weight ratios, the DNA is completely complexed by the polymers. In general, the nanoparticle diameters range from 100 nm to 200 nm, with the range for the select polymers shown from 100 – 150 nm.

Figure 3. A GFP-based secondary screen identifies several PBAE polymers that can deliver genes to adherent and suspension SCLC cell lines at efficiencies comparable to commercially available reagents

(A–B) H446 cells were transfected with PBAEs complexed with CMV-GFP DNA then analyzed by microscopy and flow cytometry. (C) The transfection efficiency and geometric mean fluorescence of 15 PBAE polymers are shown; the PBAE polymers are indicated across the x-axis. Percent transfection is presented as the mean +/− SEM of triplicate runs and the geometric mean fluorescence is the geometric mean FL1 signal +/− SEM of triplicate runs. Fugene HD was used as a control. Statistical significance of geometric mean fluorescence was determined by one-way ANOVA comparing each group with Fugene-HD (*: p ≤ 0.01). (D–F) Two SCLC suspension cell lines, H146 and H187 cells were transfected with 456:CMV-GFP and analyzed by fluorescence microscopy and flow cytometry.

Figure 4. Delivery of p53-GFP by 456 polymers induces morphologic changes, p21 expression, apoptosis and sub-G1 accumulation in bulk transfected cells

(A–B) H446 cells were transfected with 456 polymers complexed with either CMV-GFP plasmid or a CMV-p53-GFP and visualized by microscopy (A) and flow cytometry (B). (C) Percent transfection is presented as the mean +/− SEM of triplicate runs. (D) Cells were harvested at serial time points after transfection; lysates were examined for p53-GFP, p53 and p21 expression using anti-p53, anti-p21, anti-GFP (to visualize p53-GFP) antibodies. Actin was used as a loading control. (E) Annexin V staining was performed at 48 hours after transfection, cisplatin treated cells were used as a positive control for the assay. (F) Cell cycle analysis at 72 hours post-transfection.

Figure 5. Cells sorted for p53-GFP expression exhibit marked morphologic changes, p21 induction and accumulation in sub-G1 consistent with functional p53 activity

(A) H446 cells were transfected with 456 polymers complexed with either CMV-GFP or CMV-p53-GFP and sorted for GFP positive and negative populations (populations 2–5) and plated in fresh media. Untreated, sorted cells were used as a control for changes due to the sorting procedure (population 1). (B) GFP expression was quantitated by flow cytometry immediately post-sort (20H), 48 and 72 hours post-transfection. (C) Western analysis confirmed p53-GFP expression and p21 induction primarily in the p53-GFP positive (4) population. (D–E) Morphologic changes were observed by brightfield microscopy and cell cycle analysis demonstrated accumulation of cells in sub-G1. Statistical significance was determined by one-way ANOVA comparing all groups at 48 and 72 hours (***: p ≤ 0.0001 population 4 vs. 2 or 1)

Figure 6. Intratumoral delivery of 457:CMV-p53-LUC causes tumor growth inhibition

Nude mice bearing H446 xenografts received twice weekly IT injections of 457:CMV-LUC nanoparticles (n=3), 457:CMV-p53-LUC nanoparticles (n=4), or neither agent (n=3). Tumors were measured twice weekly. Group means and SEM were calculated and are shown. (A) IT injection of 457:CMV-p53-LUC resulted in statistically significant tumor growth inhibition compared with controls. (B) Area under the tumor growth curve by day 18. Statistical significance was determined by two-way ANOVA at day 18 (**: p ≤ 0.001 CMV-p53-LUC vs. CMV-LUC; ^#: p ≤ 0.01 CMV-p53-LUC vs. untreated).