Nanoformulation of Fullerene Using an Anionic Phospholipid

Abstract

In this work, fullerene was dispersed with anionic phospholipids of 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG) and small-sized fullerene nanoparticles were successfully prepared.

Fullerenes are large carbon cage molecules considered to be three-dimensional analogues of benzene. Buckminsterfullerene (C60) is the most abundant form, with 60 carbon atoms in the spherical structure (Figure 1). In fullerene, there are two types of bonds: C5–C6 double bonds in the hexagons and C5–C5 single bonds in the pentagons. So far, C60 has been frequently utilized in solar cell applications.^1-3 However, the unique physical and chemical features of C60 have recently received particular attention in the field of biomaterials. Many fullerene-based compounds with different biological activities have been synthesized for either antimicrobial or anticancer therapy, cytoprotection, enzyme inhibition, contrast- or radioactivity-based diagnosis, and controlled drug delivery.^4-11 Although ways to disperse fullerenes in water play an important role in their application in biomaterials, it is still a challenge to disperse them due to their high crystallinity. Among dispersants, phospholipids, surfactants derived from biomolecules, are promising, and the preparation of fullerene nanoparticles using DPPC, a neutral phospholipid, has been reported. ¹⁰ In order to realize wider applications of fullerene nanoparticles, it is often necessary to create particles that are controlled to smaller sizes. For example, when designing skin care materials using fullerene antioxidants, preparations of smaller fullerene nanoparticles are required to effectively penetrate the stratum corneum having high barrier properties. In this study, we report that C60 fullerenes can be effectively dispersed with an anionic phospholipid of DPPG (Figure 1) as a dispersant.

Figure 1.

Molecular structures of phospholipids of DPPG, DPPC and fullerene used in this work.

So far, we have reported that DPPG can be used to create small-sized nanoparticles encapsulating bioactive molecules such as resveratrol and paclitaxel.^12,13 As an extension of these studies, we found that DPPG can also disperse fullerene, which is difficult to disperse due to its high crystallinity. The fullerene nanoparticles can be easily prepared by mixing with DPPG in water and sonicating them. As a typical method, DPPG powder (5.0%, w/w) and fullerene powder (0.1%, w/w) were dispersed in water, and sonicated at 100 W for 2 min, and then cooled to room temperature. Visual observation of the prepared sample showed that the precipitated fullerene powder turned into a brown transparent aqueous solution with dispersed fullerenes after the mixing with DPPG. In contrast, when DPPC was used in the same way, a cloudy solution was observed. This suggests that DPPC forms a large particle in water and the fullerene is not well dispersed in the solution. We then evaluated the particle size of the particles by dynamic light scattering (DLS) analysis. When the fullerenes were observed using DPPG, a peak was observed at 19 nm, confirming that extremely small nanoparticles were created. On the other hand, particles with a larger diameter of about 50 nm were mainly observed when DPPC was used instead of DPPG, suggesting that larger particles were formed, which is in good agreement with the results of the visual observation. Phase contrast microscopy showed a homogeneous image was observed in the solution of fullerene nanoparticles with DPPG (Figure 2a), whereas large vesicles were dispersed in the samples of fullerene nanoparticles with DPPC (Figure 2b).

Figure 2.

Phase contrast microscopy of the solutions of fullerene dispersed with DPPG (a) and DPPC (b).

In addition, we compared the size of the nanoparticles in this study to those reported previously. As a typical example of fullerene nanoparticles using phospholipids, Ikeda et al reported fullerene nanoparticles using DMPC. ⁹ In this case, the particle size of the nanoparticles was about 145 nm. In addition, Miwa et al reported fullerene nanoparticles using a composite phospholipid of DPPC and DOPS, and the particle size was about 110 nm. ¹⁰ Furthermore, Miwa et al also reported fullerene nanoparticles of about 76 nm using DPPC. ¹¹ As an example of fullerene nanoparticles using a non-phospholipid surfactant, Chignell et al reported a cyclodextrin-fullerene composite with a diameter of about 20 nm. ⁸ Compared with these examples, the particle size of DPPG-fullerene was about 20 nm, which is the smallest of these examples even including the example using non-phospholipid surfactant.

We herein successfully prepared fullerene nanoparticle using anionic DPPG phospholipid. Small-sized fullerene nanoparticles were prepared compared with commonly used neutral DPPC phospholipid. Since fullerene is a promising material for ultraviolet absorption because of its high antioxidant activity, the small-sized fullerene nanoparticles in this study would be applicable to skin care materials that efficiently permeate the barrier function of the stratum corneum.

Experimental

General

Ultrasonication was performed using a QSonica model ultrasonic homogenizer. Particle sizes were measured with a Horiba model LA-960 SALD. DPPG and DPPC were purchased from Avanti Polar Lipids, and fullerene from TCI.

Preparation of Fullerene Nanoparticles

For the preparation of DPPG-fullerene, fullerene (0.1%, w/w) was mixed with DPPG powder (5.0%, w/w) in water and sonicated for 2 min to disperse homogeneously; when the mixture was heated above the T_m of DPPG the solution turned clear. The resulting mixture was kept stand at room temperature for 1 h. DPPC-fullerene was prepared in the same way, except for using DPPC powder (5.0%, w/w) instead of DPPG powder (5.0%, w/w).