Fullerenes and their derivatives have been proposed as free radical scavengers (1
), and a number of investigations have studied fullerene (C60
) derivatives as potential free radical antioxidant therapeutics (2
). Fullerenes (both pristine and derivatized fullerenes) have a tendency toward aggregation in aqueous environments making them unsuitable for therapeutic applications. For example, formulation techniques for preparing fullerene-based therapeutic candidates include host-guest complexation with cyclodextrins and calixerenes, surfactant solubilization with Tween-20 and polyvinylpyrrolidone (PVP), etc. These preparations have their respective limitations in terms of uniformity of formulation, loading capacity, aggregation and partition coefficient (5
). Derivatization of fullerenes by directly adding moieties to the carbon cage has been used as a strategy to produce useful drug candidates. Such fullerene compounds including polyhydroxylated C60
), polysulfonated C60
), carboxylated fullerenes (9
) have been shown to block free radical damages in several oxidative stress-related diseases including ischemia/reperfusion injury, inflammatory apoptosis, and neurogenerative diseases. However, aggregation in aqueous media to form particles with a broad range of diameter distributions is a general problem for many of those compounds (11
). Poly-derivatized fullerenes are often a mixture of many compounds, poorly characterized and not suitable for pharmaceutical development. In addition, it has been shown that cytotoxicity of fullerenes is related to the degree of cage derivatization, water solubility, aggregation and particle size (11
). An alternative approach in which fullerenes are encapsulated in bilayer vesicles such as liposomes has been proposed to overcome these limitations. Bensasson described the preparation of vesicles by incorporating C60
into L-α-phophatidyl cxholine purified from egg yolk (Egg-PC) (12
). However, the authors reported that only 3% or less C60
was incorporated in Egg-PC liposomes and the preparation was not uniformly reproducible. Incorporation of C60
into L-α-phophatidyl ethanolamine (PE) was limited to 7% (13
). Fullerene liposomes have also been prepared by transferring fullerenes from their water soluble host-guest complexes (C60
·γ-CD and C70
·γ-CD) to lipid membranes for photodynamic therapy (PDT) (14
). The limited number of reports on liposomal fullerene formulations was all intended to use underivatized fullerenes that are not lipophilic, rather aromatic, and structurally incompatible with natural phospholipids, therefore the fullerene contents were low and their dimensional stabilities were problematic. Further, the loading capacity of lipophilic drugs physically entrapped in liposome bilayer is limited due to the membrane destabilization effect. Thus, it is necessary to develop new drug delivery strategies that can efficiently deliver fullerenes for therapeutic applications.
Given the low contents of pristine fullerenes in liposomal formulations, we hypothesized that incorporating amphiphilic fullerenes in vesicles would greatly increase their ability to be intercalated within the lipid bilayers. Herein, we report the design and synthesis of this new class of amphiphilic fullerenes, their liposome formulation and biological activities as radical scavengers. A key to obtaining a uniform vesicular preparation with high fullerene content and dimensional stability is to incorporate amphiphilic fullerene derivatives which mimic the structure of natural phospholipids. Hirsch has reported amphiphilic C60
derivatives in which multiple aliphatic hydrocarbons were attached at various sites on the fullerene cage (16
). However these “buckysomes” don’t assemble into stable bilayers readily and the addition of multiple groups could significantly damage its bioactivities.
illustrates the design strategy in which the amphiphilic fullerenes and phospholipids are hypothesized to coassemble and form bilayer vesicles. As shown below, this method leads to highly increased loading capacity of fullerenes. The amphiphilic fullerene compounds don’t form bilayer vesicles by themselves, but require membrane-forming lipids with lipid-to-fullerene molar ratio greater than 1:1 in order to produce uniform and dimensionally stable vesicles. We have also discovered that the oval structure of C70
molecule (as opposed to the spherical C60
) provides a novel structural platform to prepare this new type of amphiphilic fullerenes. C70
has two reactive poles and a relatively inert equatorial region and this allows for sequentially attaching lipophilic and hydrophilic groups at the two poles, respectively (18
). The large underivatized zone around the C70
belt has very high radical reactivity due to the significant orbital overlap of its lowest unoccupied molecular orbital (LUMO) and highest occupied molecular orbital (HOMO) that is expected for sites of maximum ). radical reactivity (19–21 The fullerene-enriched liposome provides a novel formulation approach that not only enhances the fullerene delivery efficiency but also maintain their antioxidative properties.
Vesicle formation of amphiphilic fullerenes with auxiliary phospholipids.