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

Pharmaceutical Research volume 32, pages 2548–2558 (2015)Cite this article

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 5000:10,000, 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 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 h), and improved drug stability at pH 5.0. The IC50 of drug formulations against 4T1 cells ranged from 1.4 to 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.

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Abbreviations

BW:

Body weight

CMC:

Critical micelle concentration

DLS:

Dynamic light scattering

DMEM:

Dulbecco’s modified eagle medium

EPR:

Enhanced permeability and retention

FBS:

Fetal bovine serum

geranial:

trans-citral

i.v.:

Intravenous

LE:

Loading efficiency

MTD:

Maximum tolerated dose

neral:

cis-citral

NP:

Nanoparticle

O/W:

Oil-in-water

PBS:

Phosphate buffered saline

PCL:

Polycaprolactone

PDI:

Polydispersity index

PEG-b-PCL:

Poly(ethylene glycol)-block-polycaprolactone

SDS:

Sodium dodecyl sulfate

References

  1. Maswal M, Dar AA. Inhibition of citral degradation in an acidic aqueous environment by polyoxyethylene alkylether surfactants. Food Chem. 2013;138(4):2356–64.

    Article  CAS  PubMed  Google Scholar 

  2. Kimura K, Nishimura H, Iwata I, Mizutani J. Deterioration mechanism of lemon flavor. 2. Formation of off-odor substances arising from citral. J Agric Food Chem. 1983;31:801–4.

    Article  CAS  Google Scholar 

  3. Onawunmi GO. Evaluation of the antimicrobial activity of citral. Lett Appl Microbiol. 1989;9:105–8.

    Article  CAS  Google Scholar 

  4. Korenblum E, de Vasconcelos Goulart FR, de Almeida Rodrigues I, Abreu F, Lins U, Alves PB, et al. Antimicrobial action and anti-corrosion effect against sulfate reducing bacteria by lemongrass (Cymbopogon citratus) essential oil and its major component, the citral. AMB Express. 2013;3(1):44.

    Article  PubMed Central  PubMed  Google Scholar 

  5. Machado M, Pires P, Dinis AM, Santos-Rosa M, Alves V, Salgueiro L, et al. Monoterpenic aldehydes as potential anti-Leishmania agents: activity of Cymbopogon citratus and citral on L. infantum, L. tropica and L. major. Exp Parasitol. 2012;130(3):223–31.

    Article  CAS  PubMed  Google Scholar 

  6. Khan MS, Malik A, Ahmad I. Anti-candidal activity of essential oils alone and in combination with amphotericin B or fluconazole against multi-drug resistant isolates of Candida albicans. Med Mycol. 2012;50(1):33–42.

    Article  CAS  PubMed  Google Scholar 

  7. Helal GA, Sarhan MM, Abu Shahla AN, Abou El-Khair EK. Antimicrobial activity of some essential oils against microorganisms deteriorating fruit juices. Mycobiology. 2006;34(4):219–29.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Dudai N, Weinstein Y, Krup M, Rabinski T, Ofir R. Citral is a new inducer of caspase-3 in tumor cell lines. Planta Med. 2005;71(5):484–8.

    Article  CAS  PubMed  Google Scholar 

  9. Liu Y, Whelan RJ, Pattnaik BR, Ludwig K, Subudhi E, Rowland H, et al. Terpenoids from Zingiber officinale (Ginger) induce apoptosis in endometrial cancer cells through the activation of p53. PLoS One. 2012;7(12):e53178.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Liang CP, Wang M, Simon JE, Ho CT. Antioxidant activity of plant extracts on the inhibition of citral off-odor formation. Mol Nutr Food Res. 2004;48(4):308–17.

    Article  CAS  PubMed  Google Scholar 

  11. Yang X, Tian H, Ho CT, Huang Q. Inhibition of citral degradation by oil-in-water nanoemulsions combined with antioxidants. J Agric Food Chem. 2011;59(11):6113–9.

    Article  CAS  PubMed  Google Scholar 

  12. Djordjevic D, Cercaci L, Alamed J, McClements DJ, Decker EA. Chemical and physical stability of citral and limonene in sodium dodecyl sulfate-chitosan and gum arabic-stabilized oil-in-water emulsions. J Agric Food Chem. 2007;55(9):3585–91.

    Article  CAS  PubMed  Google Scholar 

  13. Park S, Hong C, Choi S. Citral degradation in micellar structures formed with polyoxyethylene-type surfactants. Food Chem. 2015;170:443–7.

    Article  CAS  PubMed  Google Scholar 

  14. Choi SJ, Decker EA, Henson L, Popplewell LM, McClements DJ. Inhibition of citral degradation in model beverage emulsions using micelles and reverse micelles. Food Chem. 2010;122:111–6.

    Article  CAS  Google Scholar 

  15. Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J Control Release. 2000;65(1–2):271–84.

    Article  CAS  PubMed  Google Scholar 

  16. Woodward SC, Brewer PS, Moatamed F, Schindler A, Pitt CG. The intracellular degradation of poly(epsilon-caprolactone). J Biomed Mater Res. 1985;19(4):437–44.

    Article  CAS  PubMed  Google Scholar 

  17. Kwon GS, Yokoyama M, Okano T, Sakurai Y, Kataoka K. Biodistribution of micelle-forming polymer-drug conjugates. Pharm Res. 1993;10(7):970–4.

    Article  CAS  PubMed  Google Scholar 

  18. Soo P, Luo L, Maysinger D, Eisenberg A. Incorporation and release of hydrophobic probes in biocompatible polycaprolactone-block-poly(ethylene oxice) micelles: implications for drug delivery. Langmuir. 2002;18:9996–10004.

    Article  CAS  Google Scholar 

  19. Park Y, Lee J, Chang Y, Jeong J, Chung J, Lee M, et al. Radioisotope carrying polyethylene oxide-caprolactone copolymer micelles for targetable bone imaging. Biomaterials. 2003;23:873–9.

    Article  Google Scholar 

  20. Liu J, Zeng F, Allen C. In vivo fate of unimers and micelles of a poly(ethylene glycol)-block-poly(caprolactone) copolymer in mice following intravenous administration. Eur J Pharm Biopharm. 2007;65(3):309–19.

    Article  CAS  PubMed  Google Scholar 

  21. Savic R, Azzam T, Eisenberg A, Maysinger D. Assessment of the integrity of poly(caprolactone)-b-poly(ethylene oxide) micelles under biological conditions: a fluorogenic-based approach. Langmuir. 2006;22(8):3570–8.

    Article  CAS  PubMed  Google Scholar 

  22. Tsuboi S, Ishii N, Sakai T, Tari I, Utaka M. Oxidation of alcohols with electrolytic manganese-dioxide - its application for the synthesis of insect pheromones. Bull Chem Soc Jpn. 1990;63(7):1888–93.

    Article  CAS  Google Scholar 

  23. Zeng S, Xiong MP. Trilayer micelles for combination delivery of rapamycin and siRNA targeting Y-box binding protein-1 (siYB-1). Biomaterials. 2013;34(28):6882–92.

    Article  CAS  PubMed  Google Scholar 

  24. Forrest ML, Won CY, Malick AW, Kwon GS. In vitro release of the mTOR inhibitor rapamycin from poly(ethylene glycol)-b-poly(epsilon-caprolactone) micelles. J Control Release. 2006;110(2):370–7.

    Article  CAS  PubMed  Google Scholar 

  25. Xiong MP, Forrest ML, Ton G, Zhao A, Davies NM, Kwon GS. Poly(aspartate-g-PEI800), a polyethylenimine analogue of low toxicity and high transfection efficiency for gene delivery. Biomaterials. 2007;28(32):4889–900.

    Article  CAS  PubMed  Google Scholar 

  26. Rosenberg B, Van Camp L, Grimley EB, Thomson AJ. The inhibition of growth or cell division in Escherichia coli by different ionic species of platinum(IV) complexes. J Biol Chem. 1967;242(6):1347–52.

    CAS  PubMed  Google Scholar 

  27. Cleare M, Hoeschele J. Studies on the antitumor activity of group VIII transition metal complexes. Part I. Platinum (II) complexes. Bioinorg Chem. 1973;2:187–210.

    Article  CAS  Google Scholar 

  28. Mai S, Muster B, Bereiter-Hahn J, Jendrach M. Autophagy proteins LC3B, ATG5 and ATG12 participate in quality control after mitochondrial damage and influence lifespan. Autophagy. 2012;8(1):47–62.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. White E. Deconvoluting the context-dependent role for autophagy in cancer. Nat Rev Cancer. 2012;12(6):401–10.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Parajuli P, Pisarev V, Sublet J, Steffel A, Varney M, Singh R, et al. Immunization with wild-type p53 gene sequences coadministered with Flt3 ligand induces an antigen-specific type 1T-cell response. Cancer Res. 2001;61(22):8227–34.

    CAS  PubMed  Google Scholar 

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ACKNOWLEDGMENTS AND DISCLOSURES

This research was supported by NIH grant R01DK099596 and startup funds from the University of Wisconsin-Madison, School of Pharmacy.

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Authors and Affiliations

  1. School of Pharmacy, University of Wisconsin–Madison, Madison, Wisconsin, 53705-2222, USA

    San Zeng & May P. Xiong

  2. Department of Obstetrics and Gynecology, University of Wisconsin–Madison, Madison, Wisconsin, 53705-2222, USA

    Arvinder Kapur & Manish S. Patankar

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  1. San Zeng
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  2. Arvinder Kapur
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  4. May P. Xiong
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Correspondence to May P. Xiong.

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Zeng, S., Kapur, A., Patankar, M.S. et al. Formulation, Characterization, and Antitumor Properties of Trans- and Cis-Citral in the 4T1 Breast Cancer Xenograft Mouse Model. Pharm Res 32, 2548–2558 (2015). https://doi.org/10.1007/s11095-015-1643-0

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  • DOI: https://doi.org/10.1007/s11095-015-1643-0

KEY WORDS

  • breast cancer
  • citral
  • geranial
  • micelle
  • neral