Biochemical evaluation of the anticancer potential of the polyamine-based nanocarrier Nano11047

Abstract

Synthesizing polycationic polymers directly from existing drugs overcomes the drug-loading limitations often associated with pharmacologically inert nanocarriers. We recently described nanocarriers formed from a first-generation polyamine analogue, bis(ethyl)norspermine (BENSpm), that could simultaneously target polyamine metabolism while delivering therapeutic nucleic acids. In the current study, we describe the synthesis and evaluation of self-immolative nanocarriers derived from the second-generation polyamine analogue PG-11047. Polyamines are absolutely essential for proliferation and their metabolism is frequently dysregulated in cancer. Through its effects on polyamine metabolism, PG-11047 effectively inhibits tumor growth in cancer cell lines of multiple origins as well as in human tumor mouse xenografts. Promising clinical trials have been completed verifying the safety and tolerance of this rotationally restricted polyamine analogue. We therefore used PG-11047 as the basis for Nano11047, a biodegradable, prodrug nanocarrier capable of targeting polyamine metabolism. Following exposure of lung cancer cell lines to Nano11047, uptake and intracellular degradation into the parent compound PG-11047 was observed. The release of PG-11047 highly induced the polyamine catabolic enzyme activities of spermidine/spermine N1-acetyltransferase (SSAT) and spermine oxidase (SMOX). By contrast, the activity of ornithine decarboxylase (ODC), a rate-limiting enzyme in polyamine biosynthesis and a putative oncogene, was decreased. Consequently, intracellular levels of the natural polyamines were depleted concurrent with tumor cell growth inhibition. This availability of Nano11047 as a novel drug form and potential nucleic acid delivery vector will potentially benefit and encourage future clinical studies.

Fig 1. Illustration depicting the mechanism of action for polyamine nanocarriers and their effects on polyamine metabolism.

Following endocytosis, Nano11047 undergoes thiolytic reduction by glutathione (GSH), followed by disassembly and release of PG-11047. Effects of intracellular PG-11047 accumulation on polyamine metabolism are indicated in green for upregulation or red for downregulation, resulting in an overall decrease in the natural polyamine pools required to sustain cancer cell proliferation. Abbreviations used in the figure are as follows: ODC, ornithine decarboxylase; OAZ, ODC antizyme; 3-AAP, 3-acetaminopropanal; 3-AP, 3-aminopropanal; PAOX, polyamine oxidase; SSAT, spermidine/spermine N¹-acetyltransferase; SMOX, spermine oxidase.

Fig 2. Synthesis of Nano11047.

Fig 3. ¹H-NMR of Nano11047 in D₂O.

Fig 4. Degradation of Nano11047.

Degradation of Nano11047 and PG-11047 release kinetics in 0.1 M phosphate-buffered D₂O/acetone-d6 (3/2, v/v, 0.9 mL, pH7.4) with 100 mM DTT at 25°C.

Fig 5. Cell viability in response to Nano11047.

H157 (A) or A549 (B) NSCLC cells were treated for 96 hours with increasing concentrations of Nano11047 (■) or PG-11047 (●). Cell number was determined by hemacytometer following trypan blue exclusion. Data points represent the means with error bars indicating SEM (n = 2 experiments, each performed in duplicate).

Fig 6. Nano11047 induces spermidine/spermine N¹-acetyltransferase (SSAT) activity in H157 cells.

For qRT-PCR (A), cells were treated 48 hours as indicated. For SSAT enzyme activity determination (B) and Western blot analysis (C), incubation time was increased to 72 h. Column heights indicate the means with error bars indicating SEM. *p < 0.05; **p < 0.005 determined by Student’s t-test, relative to untreated cells (n ≥ 2 experiments, each with triplicate determinations).

Fig 7. Nano11047 induces spermine oxidase (SMOX) activity.

A549 cells were treated with increasing concentrations of the analogues for 48 h. SMOX mRNA expression was determined by qRT-PCR (A); SMOX activity (B) was determined based on spermine-specific production of H₂O₂. A representative Western blot (C) indicates induction of SMOX isoforms. Column heights indicate the means with error bars indicating SEM. *p < 0.05; **p < 0.005 determined by Student’s t-test, relative to untreated cells (n ≥ 2 experiments, each with triplicate determinations).

Fig 8. Nano11047 down-regulates ornithine decarboxylase (ODC) activity.

H157 cells were treated as indicated for 72 h, and ODC enzyme activity was determined through the decarboxylation of radiolabelled ornithine. Column heights indicate the means with error bars indicating SEM. *p < 0.05; **p < 0.005 determined by Student’s t-test, relative to untreated cells (n = 2 experiments with triplicate determinations).