Formulation, Characterization, and Antitumor Properties of Trans- and Cis-Citral in the 4T1 Breast Cancer Xenograft Mouse Model

Abstract

Purpose Citral is composed of a random mixture of two geometric stereoisomers geranial (trans-citral) and neral (cis-citral) yet few studies have directly compared their in vivo antitumor properties. A micelle formulation was therefore developed. Methods Geranial and neral were synthesized. Commercially-purchased citral, geranial, and neral were formulated in PEG-b-PCL (block sizes of 5000:10000, Mw/Mn 1.26) micelles. In vitro degradation, drug release, cytotoxicity, flow cytometry, and western blot studies were conducted. The antitumor properties of drug formulations (40 mg/kg and 80 mg/kg based on MTD studies) were evaluated on the 4T1 xenograft mouse model and tumor tissues were analyzed by western blot. Results Micelles encapsulated drugs with >50% LE at 5-40% drug to polymer (w/w), displayed sustained release (t1/2 of 8-9 hours), and improved drug stability at pH 5.0. The IC50 of drug formulations against 4T1 cells ranged from 1.4-9.9 μM. Western blot revealed that autophagy was the main cause of cytotoxicity. Geranial at 80 mg/kg was most effective at inhibiting tumor growth. Conclusions Geranial is significantly more potent than neral and citral at 80 mg/kg (p<0.001) and western blot of tumor tissues confirms that autophagy and not apoptosis is the major mechanism of tumor growth inhibition in p53-null 4T1 cells.

Modulation of oxidative stress and subsequent induction of apoptosis and endoplasmic reticulum stress allows citral to decrease cancer cell proliferation

“…In these in vivo experiments, while there was significant reduction in tumor volume, even high doses of nanoparticles loaded with citral, neral or geranial did not cause noticeable toxicity in the animals57. Overall, all of these previous studies have suggested that citral and its constituents, neral and geranial, be considered as cytotoxic agents for the treatment of solid tumors.…”

“…Recently7, we demonstrated that citral and its constituent isomers neral and geranial, when administered in a nanoparticle micelle formulation, caused significant decrease in growth of 4T1 tumors in autologous BALB/c mice. In this previous study, four injections of the monoterpene formulations were administered every third day after the tumors had attained a size of 50 mm 3 .…”

“…By triggering de novo synthesis of GSH, citral could act as an anti-oxidant and hence serve an important purpose in chemoprevention. We are investigating nanoparticle-based delivery7 as well as chemical modifications as strategies to improve the pharmacokinetic profile of citral with the primary goal to develop a novel citral-based strategy for chemotherapy. The nanoparticle-citral formulations have shown increased anti-tumor efficacy in in vitro as well as in vivo experiments7.…”

“…We are investigating nanoparticle-based delivery7 as well as chemical modifications as strategies to improve the pharmacokinetic profile of citral with the primary goal to develop a novel citral-based strategy for chemotherapy. The nanoparticle-citral formulations have shown increased anti-tumor efficacy in in vitro as well as in vivo experiments7. Targeting citral-based therapeutics to tumors that have retained the ability of p53 to induce apoptosis will be an important approach to consider in future translational and clinical studies.…”

“…In this same study we showed that of the two isomers, geranial was more effective in controlling in vivo tumor growth. Retro-orbital injection of nanoparticles containing geranial at three doses of 80 mg/kg resulted in approximately 92% reduction in tumor volume as compared to controls that received unloaded nanoparticles7.…”