Felipe F. SperandioAsheesh GuptaMin WangRakkiyappan ChandranMagesh SadasivamYing-Ying HuangLong Y. ChiangMichael R. Hamblin

Photodynamic Therapy Mediated by Fullerenes and their Derivatives

BioMeDical & NaNoMeDical TechNologies – coNcise MoNograPh series

Photodynamic Therapy M

ediated by Fullerenes and their Derivatives

Scope This concise monograph series focuses on the implementation of various engineering principles in the conception, design, development, analysis and operation of biomedical, biotechnological and nanotechnology systems and applications. Authors are encouraged to submit their work in the following core topics, but authors should contact the commissioning editor before submitting a proposal:

BIoMeDIcAL DeVIceS & MATeRIALS Trauma Analysis Vibration and Acoustics in Biomedical Applications Innovations in Processing, Characterization and

Applications of Bioengineered MaterialsViscoelasticity of Biological Tissues and Ultrasound

Applications Dynamics, and Control in Biomechanical Systems Clinical Applications of Bioengineering Transport Phenomena In Biomedical Applications Computational Modeling and Device Design Safety and Risk Analysis of Biomedical Engineering Modeling and Processing of Bioinspired Materials

and Biomaterials

NANoMeDIcAL DeVIceS & MATeRIALS Bio Nano Materials Nano Medical Sciences Materials for Drug & Gene Delivery Nanotechnology for Central Nervous System Nanomaterials & Living Systems Interactions Biosensing, Diagnostics & Imaging Cancer Nanotechnology Micro & Nano Fluidics Environmental Health & Safety Soft Nanotechnology & Colloids

BioMeDical & NaNoMeDical TechNologies – coNcise MoNograPh series

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Table of Contents

1. Introduction to fullerenes and PDT 1 1.1 Introduction to fullerenes and PDT 1 1.2 Fullerenes as PS 32. Synthesis of fullerene derivatives 5 2.1 Synthesis of monocationic and polycationic

fullerene derivative examples 5 2.2 Synthesis of hexaanionic fullerene derivatives 7 2.3 Synthesis of chromophore-linked fullerene derivatives 93. Photochemistry and photophysics of fullerenyl

molecular micelle and chromophore-fullerene conjugates 104. Antimicrobial PDT mediated by fullerenes and their derivatives 12 4.1. Antimicrobial PDT mediated by

fullerenes and their derivatives 12 4.2 Fullerenes interaction with cell membrane 14 4.3 Anti-bacterial PDT with fullerenes and derivatives 15 4.4 Anti-fungal PDT with fullerenes and derivatives 22 4.5 Anti-viral PDT with fullerenes and derivatives 23 4.6 Toxicity studies of fullerenes and derivatives 245. Anti-cancer PDT 25 5.1 Photodynamic therapy of cancer cells 256. In vivo PDT studies with fullerenes 33 6.1 In vivo applications-PDT for infections and tumors 337. Conclusions & executive summary 36 7.1 Conclusion & future and perspectives 36 7.2 Executive summary 36References 39

Abstract

The fullerene molecule with its unique structure of 60 carbon atoms ar-ranged in a soccer ball structure is a molecule of great potential for a variety of applications and has drawn attention of lots of physicists, chemists and engineers. recently, these nanostructures have also been studied for their biological activities with a view towards using them for biomedical applica-tions. One of the possible therapies for which fullerenes may have a real medical application is the light based therapy called photodynamic therapy (PDT), which is a non-surgical, minimally invasive approach that has been used in the treatment of solid tumors and many non-malignant diseases.

1. Introduction to fullerenes and PDT

1.1 Introduction to fullerenes and PDTThe fullerene molecule (originally Buckminsterfullerene) was first discov-ered by Kroto et al. in 1985 and is composed of 60 carbon atoms arranged in a soccer ball-shaped structure (C60) [1]. Fullerenes have a molecular di-ameter of the order of 1 nm, however they are generally accepted as nano-particles. Their use has been rapidly increasing in the nanotechnology field, especially in engineering and physics. Polymer solar cells, for instance, are based on conjugated polymers, and fullerene derivatives can be very attrac-tive because of their mechanical flexibility as lubricants, simple fabrication process and the potential for their low-cost due to large-scale manufacture [2–4].

The fullerene molecule with its unique structure of 60 carbon atoms ar-ranged in a soccer ball structure is a molecule of great potential for a variety of applications and has drawn attention of lots of physicists, chemists and engineers. Recently, these nanostructures have also been studied for their biological activities with a view towards using them for biomedical applica-tions. One of the possible therapies for which fullerenes may have a real medical application is the light based therapy called photodynamic therapy (PDT) [5], which is a non-surgical, minimally invasive approach that has been used in the treatment of solid tumors and many non malignant dis-eases [6]. PDT is a non-thermal photochemical reaction, which requires the simultaneous presence of a photosensitizing drug (photosensitizer, PS), oxygen and visible light (Figure1-1). It is a two-step procedure that involves the administration of a PS, followed by activation of the drug with the ap-propriate wavelength of light [7–10]. The photoactivation of the drug gen-erates singlet oxygen and other reactive oxygen species (ROS), which cause a lethal oxidative stress and membrane damage in the treated cells and in the case of tumors, leads to cell death by direct cytotoxicity and a dramatic anti-vascular action that impairs blood supply to the area of light exposure [11]. It is known that depending on the parameters involved, PDT in vitro can kill cancer cells via apoptosis, necrosis or autophagy. The direct killing effect of PDT on malignant cancer cells has been studied in detail in vitro, and also clearly applies in vivo; but in addition, two separate in vivo mechanisms leading to PDT-mediated tumor destruction have been described. They are the vascular shutdown effect mentioned above [12] and the PDT-induced activation of the host immune system [13].

The most common chemical structures that have been employed as PS for PDT purposes are derived from the tetrapyrrole aromatic nucleus found in many naturally occurring pigments such as heme, chlorophyll and bacterio-chlorophyll. Tetrapyrroles usually have a relatively large absorption band in the region of 400-nm known as the Soret band, and a set of progressively smaller

2 Photodynamic Therapy Mediated

absorption bands as the spectrum moves into the red wavelengths known as the Q-bands. Another broad class of potential PS includes completely synthetic, non-naturally-occurring, conjugated pyrrolic ring systems. These comprise such structures as texaphyrins [14], porphycenes [15], and phthalocyanines [16]. Other compounds that have been studied as PS are non-tetrapyrrole de-rived dyes, that may be either naturally occurring or totally synthetic, and these compounds have often been used as antimicrobial PS. Examples of the first group (naturally occurring dyes) are hypericin [17] and from the second group (synthetic dyes) are toluidine blue O [18] and Rose Bengal [19].

Some of the characteristics that the ideal PS should possess are the pres-ence of low levels of dark toxicity and presence of absorption bands that should be in the so called optical window (600‒900nm) for sufficient tis-sue penetration of light. They should have relatively high absorption bands (>20,000‒30,000 M‒1cm‒1) to minimize the dose of PS needed to achieve the desired effect. PS should ideally have high triplet and singlet oxygen quantum yields. The usefulness of various PS proposed for antimicrobial PDT has to be judged on different criteria. One of the requirements is that an antimicrobial PS should be able to kill multiple classes of microbes at relatively low concentrations and low fluences of light. PS should be reason-ably non-toxic in the dark and should show selectivity for microbial cells over mammalian cells. Up to the present date there is no single perfect PS that meets all the features of an ideal PS. The reason why fullerenes are seen as potential PDT agents is that they possess some of the characteristics that render them well suited as a photosensitizing drug as detailed below.

Figure 1-1 Jablonski diagram showing excitation of fullerene to excited singlet state, intersystem crosssing to long-lived triplet state and Type 1 and II photochemical mechanisms to produce ROS capable of destroying DNA, viruses, pathogenic micro-organisms, tumors and other unwanted cells.

Introduction 3

1.2 Fullerenes as PSDue to rapidly growing interest in the medical application of nanotechnol-ogy, fullerenes have gained considerable attention as possible PS for PDT mediators [20]. It is known that pristine C60 is highly insoluble in water and biological media and forms nanoaggregates that prevent its efficient photoactivity [21]. However when fullerenes are derivatized by chemists who attach some functional groups to these molecules to make them more soluble in water and in biologically compatible solvents their biological/medical usefulness is markedly improved [22]. Different hydrophilic or amphiphilic side chains or fused ring structures have been attached to the spherical C60 core. This solubility imparts a higher ability to produce singlet oxygen, hydroxyl radicals and superoxide anion, and these molecules have been proposed as effective PDT mediators in several applications. Some of the advantages that these fullerenes possess over the traditional PS are:

1) Fullerenes are comparatively more photostable and demonstrate less photobleaching compared to tetrapyrroles and synthetic dyes;

2) Fullerenes show both kinds of photochemistry, comprising Type I (free radicals) and Type II (singlet oxygen), while tetrapyrroles demonstrate largely Type 2 photochemistry;

3) Fullerenes can be chemically modified for tuning the drug’s par-tition coefficient (Log P or partition coefficient for [drug in n-octanol]/[drug in H2O]) and pKa values for the variation of in vivo lipophilicity and the prediction of their distribution in bio-logical systems;

4) To enhance the overall quantum yield and the ROS production and to extend their absorption spectrum further into the red wave-lengths, a light harvesting antennae can be chemically attached to C60;

5) Molecular self-assembly of fullerene cages into vesicles allows im-proved drug delivery and can produce self-assembled nanoparti-cles that may have different tissue targeting properties.

Besides these advantages, fullerenes show some disadvantages, which can also be overcome by applying special strategies. One of the concerns for the use of fullerenes is their poor biodegradability, as these nanostructures may accumulate in the environment [23]; however, studies have concluded that C60 itself is remarkably non-toxic [24]. In fact, the lifespan of rats was found to be increased by the oral administration of C60 [25]. Another concern was their extreme hydrophobicity and their innate tendency to aggregate, which renders them less promising for application as drugs in biomedicine. Figure 1-2 illustrates some of these strategies that have been used to solubilize fullerenes. The following strategies have been applied for solubilization and drug-delivery

4 Photodynamic Therapy Mediated

of fullerenes e.g liposomes (Figure 1-2B) [26–28], micelles (Figure 1-2A) [29, 30], dendrimers (Figure 1-2E) [31, 32], PEGylation [33–36] (Figure 1-2F), cyclodextrin encapsulation [37, 38] (Figure 1-2D, and self-nanoemul-sifying systems (SNES) (Figure 1-2C) [39–42] to overcome this shortcom-ing of fullerenes. Besides these disadvantages, the main optical absorption band of fullerenes is in the blue and green regions, whereas the absorption spectra of tetrapyrrole PS other than porphyrins (such as chlorins, bacteri-ochlorins and phthalocyanines) have been designed to have substantial ab-sorption peaks in the red or far-red regions of the spectrum. In order to be useful in vivo it is considered that the light used to excite the PS should be in the red/near-infrared (NIR) spectral region where scattering and absorp-tion of light by tissue is minimized. This unfavorable absorption spectrum of fullerenes can be overcome by various strategies such as covalent attach-ment of light harvesting antennae to fullerenes [43–47], by using optical clearing agents [48–52] or by applying two-photon excitation where two NIR photons are simultaneously delivered to be equivalent to one photon of twice the energy (and half the wavelength, so that two 800-nm photons are equivalent to one 400-nm photon) [53–57].

Figure 1-2 Drug delivery vehicles that have been used to solu-bilize fullerenes.