Pharmaceutical Nanotechnology
Nanoemulsions of an anti-oxidant synergy formulation containing gamma tocopherol have enhanced bioavailability and anti-inflammatory properties

https://doi.org/10.1016/j.ijpharm.2008.07.022Get rights and content

Abstract

The present study investigated whether MicroFluidizer Processor®-based nanoemulsions of an antio-xidant synergy formulation (ASF), containing delta, alpha and gamma tocopherol influenced inflammation and bioavailability in CD-1 mice. Croton oil was applied to all animals’ right ear lobe to induce inflammation. Auricular thickness was measured after 2 and 6 h after the various treatments. The animal plasma and ear lobes were collected and frozen for bioavailability and cytokine analyses. The ASF nanoemulsions of alpha, delta, or gamma tocopherol significantly reduced auricular thickness compared to control (57, −57, and −71%, respectively) and blank nanoemulsion (−50, −50, −67%, respectively). Relative to the suspensions of ASF, only the nanoemulsion of ASF containing gamma tocopherol significantly reduced auricular thickness (−60%), whereas the 40% reduction with nanoemulsions of delta tocopherol compared to suspension was not statistically significant. Auricular concentrations of cytokines TNF-alpha and IL-1 alpha were significantly reduced in mice treated only with ASF nanoemulsions of gamma tocopherol compared to control (−53, −46%, respectively) and blank nanoemulsion (−52, −46%, respectively). Auricular thickness was significantly associated with tissue TNF-alpha (r = 0.539, p < 0.001) and IL-1 alpha concentrations (r = 0.404, p = 0.01). Bioavailability for gamma and delta was dramatically enhanced (2.2- and 2.4-folds) with the nanoemulsion compared to suspensions. Only the plasma gamma tocopherol concentration was significantly associated with auricular thickness (r = −0.643, P = 0.001). In conclusion, nanoemulsions of ASF containing gamma, alpha, and delta tocopherol, have enhanced anti-inflammatory properties and increased bioavailability, with gamma tocopherol, in particular compared to their suspensions.

Introduction

Inflammation is the body's first immune response when infected or irritated by external assault. Although inflammation is associated with the normal process of healing, the mechanism(s) involved in this process are quite complex, and when not well regulated, can result in inflammatory disease. In addition, clinical evidence has shown that chronic inflammation can contribute to the development of certain kinds of cancers (Coussens and Werb, 2002), neurodegenerative diseases (Stix, 2007) and atherosclerosis (Libby, 2002).

Vitamin E contains eight different isomers of tocopherols and tocotrienols that vary in degree of anti-oxidant and anti-inflammatory properties. All the various analogs of tocopherol and tocotrienol have similar structures on their chromanol ring with only the number of methyl groups on the chromanol ring differing between the alpha, beta, gamma, and delta isomers. In addition, the different bioavailability of each form is thought to explain the range of their biological activities and functions (Bieri and Evarts, 1974, Jiang et al., 2001, Valenzuela et al., 2002).

Alpha tocopherol is an important lipid peroxidation anti-oxidant and the predominant form of tocopherol found in mammalian circulation and tissues. Experimental data have shown that alpha tocopherol has ability to modulate several enzyme activities, such as protein kinase C, cyclooxygenase-2 (COX-2), phospholipase A2, and protein phosphatase 2A (Reiter et al., 2007, Zingg, 2007) which are involved in the signal transduction pathway of inflammation (Reiter et al., 2007). Although gamma tocopherol is the most abundant isomer in the American diet, its appearance in the blood stream (bioavailability) is limited reportedly, in part, as a result of reduced retention time of gamma tocopherol in the body when compared to alpha tocopherol (Jiang et al., 2001). Recent data from experimental studies suggest that gamma tocopherol can act as an anti-inflammatory agent by reducing the synthesis of prostaglandin E2, through the inhibition of cyclooxygenases such as COX-2 (Wolf, 2006).

Previously, our lab has reported that an anti-oxidant synergy formulation (ASF), which contained alpha tocopherol exerts synergistic effects on neuroblastoma cells by providing neuroprotection against oxidative stress, reducing cellular proliferation, and promoting differentiation in a series of in vitro experiments (Amy et al., 2003). As a result of this study, we hypothesized that (a) this ASF formulation might have other biological indications (anti-inflammation) and also whether the individual tocopherol isomers in the ASF formulation were different, as it relates to inflammatory activity (b) the bioavailability and efficacy of this tocopherol component of ASF formulation might similarly be enhanced by altering the nano-delivery system, in particular, from the self-assembly polyethylene glycol (PEG)-based polymer/solvent nanospheres reported in our initial studies of tocopherol (Shea et al., 2005) to the present nanoemulsion formulation utilizing Microfluidizer Processor® Technology. These possible outcomes were enhanced by our recent reports that agents delivered by our MicroFluidizer Processor®-based nanoemulsion system showed increased anticancer indications (Kuo et al., 2007, Tagne et al., 2008) and bioavailability (Kotyla et al., 2008).

Much attention has focused on developing alternative drug delivery systems, nanoscale, in particular, in order to improve the bioavailability of poorly water soluble or hydrophobic compounds which could lead to greater efficacy. Compared to most other formulations, emulsion preparations, composed of oil, surfactant, and water are associated with less toxicity and increase bioavailability and efficacy of the various encapsulants. For example, microemulsions containing micron-sized particles (thousands of nanometers in size) increase water solubility and bioavailability of nitrendipine, prednisolone, and betamethasone (Kawakami et al., 2002, Lawrence and Rees, 2000). A logical extension of these published reports are that, since nanoemulsions have particle sizes usually less than or slightly greater than 100 nm and have increased surface-to-volume ratio, they would be expected to significantly enhance bioavailability and efficacy as reported by others, (Kumar et al., 2004, Shafiq et al., 2007) and from our own laboratory with encapsulants consisting of such diverse compounds as ASF (Kuo et al., 2007), tamoxifen (Tagne et al., 2008), and tocopherol (Kotyla et al., 2008).

The objectives of the current study were to utilize the inflammatory CD-1 mouse model which we have previously reported on (Yoganathan et al., 2003) to (a) compare the anti-inflammatory properties of ASF containing different tocopherol isomers and (b) evaluate whether nanoemulsion formulations of ASF containing different tocopherol isomers will affect the bioavailability and efficacy compared to their control, empty nanoemulsions and suspension counterpart.

Section snippets

Suspension and nanoemulsion preparations

Alpha, delta, and gamma tocopherol isomers, sodium pyruvate, polysorbate 80 and phosphatidyl choline were purchased from Sigma–Aldrich Inc. (St. Louis, MO). Suspensions of these ingredients were prepared in the following manner: phosphatidyl choline and tocopherol isomers were dissolved in ethanol and a mixture of sodium pyruvate and HPLC-grade water were added and then homogenized for 30 s (Polytron Model PT 10/35, Brinkmann Instr., Westbury, NY). For the preparation of nanoemulsions,

Measurement of particle sizes of the ASF formulations

The various ASF formulations were subjected to dynamic laser light scattering particle size analysis. The average particle sizes of the ASF suspensions were 843.0 ± 9.2 nm, 758 ± 10.5 nm and 673 ± 13.6 nm (mean ± S.E.M.; n = 3 measurements) for alpha, delta, and gamma tocopherol, respectively) (Table 1). In comparison, for the ASF nanoemulsions containing different tocopherol isomers, the dramatically reduced average particle sizes were 56.6 ± 0.5 nm, 51.1 ± 0.5 nm and 42.3 ± 0.2 nm (mean ± S.E.M.; n = 3 measurements)

Discussion

The current study has found that particle size is a very critical factor in decreasing inflammation and increasing transdermal permeability of certain tocopherol isomers. Smaller particle size is considered to be advantageous since the interfacial area of encapsulated compounds into the aqueous phase is maximized (Pan et al., 2002, Vila et al., 2005). According to previous experiments, the results indicated that Microfluidizer Processor®-based nanoemulsion system significantly increase the

Acknowledgements

We acknowledge Animal Facility Manager Sheryl Perry for her technical assistance and Maureen Faul of the Center for Health and Disease Research for her administrative assistance. These studies were partially supported by Microfludics Corp., Newton, MA 02464

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