Nanobiomaterials in Antimicrobial Therapy: Applications of Nanobiomaterials

By Alexandru Grumezescu

Nanobiomaterials in Antimicrobial Therapy presents novel antimicrobial approaches that enable nanotechnology to be used effectively in the treatment of infections. This field has gained a large amount of interest over the last decade, in response to the high resistance of pathogens to antibiotics.

Leading researchers from around the world discuss the synthesis routes of nanobiomaterials, characterization, and their applications as antimicrobial agents. The books covers various aspects: mechanisms of toxicity for inorganic nanoparticles against bacteria; the development of excellent carriers for the transport of a high variety of antimicrobials; the use of nanomaterials to facilitate both diagnosis and therapeutic approaches against infectious agents; strategies to control biofilms based on enzymes, biosurfactants, or magnetotactic bacteria; bacterial adhesion onto polymeric surfaces and novel materials; and antimicrobial photodynamic inactivation.

This book will be of interest to postdoctoral researchers, professors and students engaged in the fields of materials science, biotechnology and applied chemistry. It will also be highly valuable to those working in industry, including pharmaceutics and biotechnology companies, medical researchers, biomedical engineers and advanced clinicians.

• A methodical approach to this highly relevant subject for researchers, practitioners and students working in biomedical, biotechnological and engineering fields
• A valuable guide to recent scientific progress and the latest application methods
• Proposes novel opportunities and ideas for developing or improving technologies in nanomedicine and nanobiology

• Language: English
• Publisher: Elsevier Science
• Release date: Mar 8, 2016
• ISBN: 9780323428873

Book preview

Ukraine

Preface of the series

Ecaterina Andronescu, Department of Science and Engineering of Oxide Materials and Nanomaterials, Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Bucharest, Romania

The era of nanosized materials is now considered the center of the evolution of future tools and emerging technologies with wide applications in industry, research, health, and beyond. Despite recent scientific progress, biological applications of nanomaterials are far from being depleted and current knowledge is limited by the poor access to significant data, but also by widespread and usually unfounded speculation. Although exhaustive, the current literature is difficult to reach and understand because of the specificity and strict focuses of researchers investigating different applications of nanomaterials.

In this context, the scientific series entitled Applications of Nanobiomaterials was motivated by the desire of the Editor, Alexandru Mihai Grumezescu, and others to bring together comprehensive, up-to-date and relevant findings on the field of biological applications of nanostructured materials, to promote the knowledge and expand our vision regarding future perspectives. Even though the approached domain is quite specific and research-oriented, this multivolume set is easily intelligible for a wide audience including: under-graduate and post-graduate students, engineers, researchers, academic staff, pharmaceutical companies, biomedical sector, and industrial biotechnologies. However, some basic knowledge of the field of materials science (nanobiomaterials, pharmaceutical industry, products for medicinal treatments, nanoarchitectonics for delivery of biological active molecules and release, bone implants, and stomatology) and engineering is a requisite for understanding technical aspects.

The selected authors of each chapter are outstanding specialists in the field of nanobiomaterials, who have made impressive contributions in a specific area of research or applied area within the scope of this book.

Each of the 11 volumes of the series contains 15 chapters, addressing the most relevant and recent matters on the field of the volume.

The first volume, Fabrication and Self-Assembly of Nanobiomaterials, introduces the reader to the amazing field of nanostructured materials and offers interesting information regarding the fabrication and assembly of these nanosized structures. In Volume II, entitled Engineering of Nanobiomaterials, readers can easily find the most commonly investigated methods and approaches for obtaining tailored nanomaterials for a particular application, especially those with a great deal of significance in the biomedical field. In the following step, readers will discover the importance and the ways of modifying the surface of nanostructured materials to obtain bioactive materials, by reading Volume III, Surface Chemistry of Nanobiomaterials. Starting with Volume IV Nanobiomaterials in Hard Tissue Engineering and Volume V Nanobiomaterials in Soft Tissue Engineering the biomedical applications of engineered nanomaterials are revealed and discussed, focusing on one of the most impacted fields, tissue engineering. Volume VI, Nanobiomaterials in Antimicrobial Therapy, highlights the potential of different nanostructured materials to be utilized in the development of novel efficient antimicrobial approaches to fight the global crisis of antibiotic inefficiency and emerging infectious diseases caused by resistant pathogens. Volume VII moves on to another key biomedical domain—cancer therapy. This volume, Nanobiomaterials in Cancer Therapy, describes current issues of cancer therapy and discusses the most relevant findings regarding the impact of nanobiomaterials in cancer management. Medical Imaging represents the focus of Volume VIII, while Volume IX deals with applications of Nanobiomaterials in Drug Delivery. Volume X, entitled Nanobiomaterials in Galenic Formulations and Cosmetics, refers to the perspectives highlighted by the utilization of nanosized functional biomaterials in the development of improved drugs and active principles for different biomedical industries. Finally, Volume XI is dedicated to the impact of Nanobiomaterials in Dentistry, which currently represents one of the most investigated and controversial domains related to the biomedical applications of nanostructured materials.

Due to their specific organization, each volume can be treated individually or as a part of this comprehensive series, which aims to bring a significant contribution to the field of research and biomedical applications of nanosized engineered materials.

Preface

Alexandru Mihai Grumezescu, http://grumezescu.com/, Department of Science and Engineering of Oxide Materials and Nanomaterials, University Politehnica of Bucharest, Bucharest, Romania, Department of Biomaterials and Medical Devices, Faculty of Medical Engineering, University Politehnica of Bucharest, Bucharest, Romania

About the Series (Volumes I–XI)

The increased fabrication of nanosized materials with applications on the biomedical field by using biomimetic and bio-inspired processes and formulations, has recently led to a new concept, nanobiotechnology. This complex research brings together significant knowledge from physical, chemical, biological, and technological sciences in an applicative field.

Medical applications of nanobiomaterials range from the development of adequate scaffolds for tissue engineering to therapeutic nanostructures, such as targeted drug delivery systems. The purpose of this multivolume set entitled Applications of Nanobiomaterials is to offer a broad, updated, and interdisciplinary point of view regarding the application of these materials of the future medicine, starting with their fabrication, specific engineering and characterization and ending with the most investigated applications such as tissue engineering, antimicrobial and cancer therapies, and also the development of different medical and cosmetic use products. These books bring together the work of outstanding contributors who have significantly enhanced the basic knowledge and applicative concepts of this research field in their respective disciplines.

The multivolume set Applications of Nanobiomaterials contains 165 chapters, organized in 11 volumes to present a novel and up-to-date approach related to this intriguing domain. Each chapter was carefully composed and illustrated to highlight the relevance of nanobiomaterials on most biomedical fields, revealing the most recent applications on a specific domain. The whole set represents a great material for the academic community, starting with undergraduate and postgraduate students, researchers, engineers, and medical doctors, but also pharmaceutical companies and innovative biotechnologies.

These 11 volumes cover all relevant aspects related to the Applications of Nanobiomaterials and as it follows:

Volume I: Fabrication and Self-Assembly of Nanobiomaterials

Volume II: Engineering of Nanobiomaterials

Volume III: Surface Chemistry of Nanobiomaterials

Volume IV: Nanobiomaterials in Hard Tissue Engineering

Volume V: Nanobiomaterials in Soft Tissue Engineering

Volume VI: Nanobiomaterials in Antimicrobial Therapy

Volume VII: Nanobiomaterials in Cancer Therapy

Volume VIII: Nanobiomaterials in Medical Imaging

Volume IX: Nanobiomaterials in Drug Delivery

Volume X: Nanobiomaterials in Galenic Formulations and Cosmetics

Volume XI: Nanobiomaterials in Dentistry

About Volume VI

Volume VI, entitled Nanobiomaterials in Antimicrobial Therapy, represents an extensive and up-to-date book regarding the current progress of nanotechnology and the science of nanobiomaterials in anti-infective therapy. The editor brings together state-of-the-art chapters describing the synthesis routes of nanobiomaterials, characterization, and their applications as antimicrobial agents. Important aspects such as: (i) mechanisms of toxicity for some inorganic nanoparticles against bacteria; (ii) the development of excellent carriers for the transport of a high variety of antimicrobials; (iii) the use of nanomaterials to facilitate both diagnosis and therapeutic approaches against infectious agents; (iv) strategies to control biofilms based on enzymes, biosurfactants, or magnetotactic bacteria; (v) bacterial adhesion onto polymeric surfaces and novel materials; and (vi) antimicrobial photodynamic inactivation are discussed.

Volume VI contains 15 chapters, prepared by outstanding international researchers from the United States of America, Brazil, Spain, Romania, Russia, Iran, India, Malaysia, and South Korea.

In Chapter 1, Antimicrobial Photoinactivation with Functionalized Fullerenes, Lucas F. de Freitas et al. give an overview about photodynamic therapy by covering the most relevant studies using functionalized fullerenes in antimicrobial photodynamic inactivation.

Roxana Cristina Popescu et al., in Chapter 2, Toxicity of Inorganic Nanoparticles Against Prokaryotic Cells, present an up-to-date review regarding the mechanisms of toxicity for some inorganic nanoparticles against bacteria. Also, some newly developed model systems are described, focusing on the biosynthesized nanoparticles, which are clearly a trend in this field.

Chapter 3, Antimicrobial Magnetosomes for Topical Antimicrobial Therapy, prepared by Revathy et al., discusses the use of magnetosomes produced by magnetotactic bacteria to penetrate the biofilm matrix. By comparison with synthetic magnetic nanoparticles, these unique magnetosomes present low toxicity, ecofriendly, and cost-efficient properties.

In Chapter 4, Synthesis, Characterization, and Applications of Nanobiomaterials for Antimicrobial Therapy, C. Ganesh Kumar et al. discuss the synthetic routes of nanobiomaterials, characterization, and applications as potential antimicrobial agents. A number of case studies on the biosynthesis of nanobiomaterials are also presented.

Juan Rodríguez-Hernández et al., in Chapter 5, Antimicrobial Micro/Nanostructured Functional Polymer Surfaces, present an up-to-date overview about bacterial adhesion onto polymeric surfaces. In particular, the chapter focuses on the parameters that are involved in the adhesion of microorganisms to polymeric surfaces including the surface chemistry or the topography at the micro/nanometer scale.

Subhashini Mohanbaba et al., in Chapter 6, Differential Biological Activities of Silver Nanoparticles Against Gram-Negative and Gram-Positive Bacteria – A Novel Approach for Antimicrobial Therapy, present the impact of silver nanoparticles utilized as alternative antibacterial nanobiotics by describing their biogenesis, characterization, mechanisms of action against bacteria, and industrial applications, along with future perspectives.

In Chapter 7, Enhancement of Pathogen Detection and Therapy with Laser-Activated, Functionalized Gold Nanoparticles, Randolph D. Glickman et al. describe the use of nanomaterials to facilitate both diagnostic and therapeutic approaches against infectious agents. The authors focused on current efforts involving the use of functionalized gold nanoparticles for diagnostic and therapeutic applications in infectious disease. The preparation, conjugation, and eventual deployment of these nanoparticle platforms are also described in this chapter.

In Chapter 8, Antimicrobial Properties of Nanobiomaterials and the Mechanism, Mashitah et al., highlight the history of nanobiomaterials utilized as antimicrobial agents and their reaction mechanism. The use of nanomaterials in food packaging, water purification, and disinfection are also discussed here.

In Chapter 9, prepared by Badal Kumar Mandal et al., Scopes of Green Synthesized Metal and Metal Oxide Nanomaterials in Antimicrobial Therapy, the authors review the main nanobiomaterials used as antimicrobial agents (silver, gold, iron, copper, iron oxide, copper oxide, zinc oxide, titanium dioxide, silver oxide, cadmium oxide, alumina, nickel oxide, silica, tin oxide, and cadmium telluride) toward various microorganisms. Also, the authors provide a brief description of other non-metallic carbon-based nanomaterials and highlight their mechanistic approach.

Farnoush Asghari et al., in Chapter 10, Antifungal Nanomaterials: Synthesis, Properties, and Applications, discuss recent advances in the field of antifungal nanomaterials from synthesis to applications in human health, environment, and food industries.

Chapter 11, Strategies Based on Microbial Enzymes and Surface-Active Compounds Entrapped in Liposomes for Bacterial Biofilm Control, by Vera Lúcia dos Santos et al., reveal the most recent strategies aiming to control biofilms based on enzymes and biosurfactants free or entrapped in liposomes.

Nadezhda M. Zholobak et al., in Chapter 12, Interaction of Nanoceria with Microorganisms, compile the existing data on the influence of nanoceria on bacteria, including clinically important bacterial strains. The mechanisms of antimicrobial action and suggested reasons behind the different sensitivities of different types of microorganisms to nanoceria are discussed.

In Chapter 13, PLA and PLGA Nanoarchitectonics for Improving Anti-Infective Drugs Efficiency, Carmen Mariana Chifiriuc et al. give an overview of PLA and PLGA polymers that proved to be excellent carriers for the transport, delivery, and controlled release of a large variety of drugs and other categories of bioactive substances (genes, peptides, proteins, antigens, vaccines, growth factors, etc.) in order to be able to select the most appropriate composition and formulation dedicated to short-term or long-term clinical applications.

Neha Sharma et al., in Chapter 14, Nanoparticles: Boon to Mankind and Bane to Pathogens, focus on different types of metallic nanoparticles as well as mechanisms of action of bactericidal and fungicidal nanoparticles.

Chapter 15, Scientometric overview regarding the nanobiomaterials in antimicrobial therapy prepared by Ozcan Konur et al., gives a scientometric overview about the research of antimicrobial nanobiomaterials, and brief information on the key stakeholders about the influential papers in this dynamic research field as the first-ever study of its kind.

Chapter 1

Antimicrobial photoinactivation with functionalized fullerenes

Lucas F. de Freitas¹,² and Michael R. Hamblin²,³,⁴,    ¹Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, São Paulo, Brazil,    ²Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA,    ³Department of Dermatology, Harvard Medical School, Boston, MA, USA,    ⁴Harvard-MIT Division of Health Science and Technology, Cambridge, MA, USA

Abstract

Photodynamic therapy (PDT) is based on the interaction between light and a nontoxic photosensitizer to produce a long-lived triplet state that reacts in the presence of oxygen, generating reactive oxygen species (ROS) through a type I reaction, or generating singlet oxygen through a type II reaction. Porphyrins and their derivatives were the first compounds to be used as photosensitizers, however, a great number of different molecules have since been shown to be effective in PDT, including fullerenes. The first fullerene, C60, was discovered in 1985, and since then the unique properties of these compounds have attracted the attention of researchers in the biomedical area. Due to their extended π-conjugation, they can absorb light in the UV and visible spectrum, and can generate ROS, especially in the presence of electron donors (mainly through a type I reaction, generating superoxide anion). The functionalization of fullerenes, in order to enhance their solubility in aqueous media and their absorption in the visible spectrum, has increased the range of biomedical applications of these compounds, and promising results have been published regarding antitumor and antimicrobial PDT in vitro and in vivo. This chapter covers the most relevant studies using functionalized fullerenes in antimicrobial photodynamic inactivation.

Keywords

Fullerenes; photodynamic inactivation; antimicrobial therapy; functionalization; molecular antennae; photosensitizers

1.1 Introduction

Albert Einstein proposed the theory behind the phenomenon that came to be known as light amplification by stimulated emission of radiation (LASER) back in 1917, but at least 40 years passed before a working laser was constructed for the first time by Theodore Maiman in 1960. In fact, in 1956 Charles Townes and Arthur Schawlow published a study entitled Infrared and optical maser, about microwave amplification by the stimulated emission of radiation (MASER), and this study served as a basis for Maiman’s work. The applications of lasers grew exponentially over the succeeding decades, including applications in medicine (Round et al., 2013).

One of the most promising applications of lasers in medicine is related to the activation of molecules, a group of dyes called photosensitizers, by light. Photodynamic therapy (PDT) is a minimally invasive approach that has broad applications, that is, as an alternative to conventional anticancer therapies such as chemotherapy and radiotherapy, and to treat many nonmalignant diseases, such as acne, macular degeneration of the retina, and for antimicrobial inactivation (Bazylińska et al., 2012; Song et al., 2014).

The photodynamic process relies on three main constituents: the photosensitizer (a molecule that has low to zero dark toxicity, and should accumulate selectively in the target tissue); light (at a specific wavelength in order to activate the photosensitizer to a higher energy state); and molecular oxygen (which will receive energy from the activated photosensitizer, generating reactive oxygen species (ROS)) (Nowak-Stępniowska et al., 2011).

PDT is a two-step process: the first step is the administration of the photosensitizer, and the second is the activation by light. Basically, the photosensitizer is activated by a harmless visible light irradiation, going from the ground singlet state (two electrons with opposite spins in the highest occupied molecular orbital (HOMO)) to a short-lived activated singlet state (one of the electrons goes to the lowest unoccupied molecular orbital (LUMO), but retains the same spin). The loss of energy from this excited electron can lead to fluorescence emission. Through a process termed intersystem crossing, the photosensitizer can go to a triplet excited state (two electrons now have parallel spin) and this state is long-lived. Its long lifetime allows it to undergo chemical reactions, such as the transfer of an electron to molecular oxygen, generating radicals such as superoxide (O2−), which leads to hydroxyl radicals (HO) through a type I reaction, or it can transfer energy to molecular oxygen, generating singlet oxygen (¹O2) through a type II reaction (Figure 1.1). The ratio between type I and type II reactions depends on the photosensitizer used and on the microenvironment surrounding it (Castano et al., 2004, 2005a,b; Huang et al., 2012). The oxygen supply is a determinant for PDT outcomes, meaning that a hypoxic environment can compromise the success of PDT.

Figure 1.1 Photodynamic therapy mechanism. The photosensitizer (PS) in a ground singlet state is excited by light to an activated singlet state (¹PS*), and after an intersystem crossing to a triplet state (³PS*) it transfers energy to molecular oxygen, generating singlet oxygen (type II reaction), or transfers electrons to other molecules (type I reaction), generating reactive species that will oxidize biomolecules on the surroundings (from microorganisms or from cancer cells).

Dual selectivity with PDT can be achieved, firstly due to the preferential accumulation of photosensitizers in the target cells (cancer cells, other cells with impaired functions, or microorganisms) or in the diseased tissues (due to abnormal blood supply), and secondly due to the spatially confined delivery of light to activate the photosensitizer (Agostinis et al., 2011).

The longer the wavelength that is used, the deeper it penetrates into the biological tissues. Therefore, for superficial conditions, such as skin conditions and wound infections, higher-energy shorter wavelengths can be used, while for deep-seated diseases like cancer it is necessary to use irradiation with longer wavelength light, usually red or near infrared (Allison et al., 2004).

1.2 Photosensitizers

Photosensitizers are molecules that can be activated by light in order to generate ROS that can damage cell structures from microorganisms or from diseased mammalian cells leading to cell death. Ideally, photosensitizers should be relatively easy to synthesize as a single pure compound, have low levels of cytotoxicity in the dark, and have no physiological side effects (such as hypotension or hypersensitivity). Moreover, photosensitizers that are activated by longer wavelengths of light, especially in the red and far red, can be used to treat sites that are deeper in the body, since the penetration of red light into tissues is higher and there is less skin photosensitivity in this region of the spectrum. They should have strong absorption bands (higher than 30,000 M−1 cm−1), so that a lower amount of light or a lower dose of photosensitizer can be used, and the drug clearance from the patient should be as rapid as possible to avoid the necessity of long-term protection from light after the treatment. Finally, the photodynamic activity of the photosensitizer should be high enough to guarantee good outcomes, but must not be too high to the point that an overdose could inadvertently occur, causing damage to normal tissue (Allison et al., 2004; Detty et al., 2000).

The first generation of photosensitizers was naturally occurring porphyrins and their derivatives. These compounds were developed in the 1970s and were at first thought to have good photodynamic activity, but were then found to have disadvantages such as prolonged cutaneous phototoxicity, low absorption bands at red wavelengths, and some dark cytotoxicity. Tetrapyrrole compounds have a strong absorption around 400 nm (the Soret band) and only much smaller absorption bands at longer wavelengths (Q-bands). Naturally occurring porphyrins are conjugated molecules with varying numbers of carboxyl groups and with absorption at wavelengths no more than 630 nm (Allison et al., 2004).

Some second-generation photosensitizers were developed in an attempt to overcome the disadvantages of first-generation compounds, and good-to-moderate success was obtained. The third-generation photosensitizers consist of second-generation photosensitizers that have been modified with targeting agents (such as antibody conjugation) or encapsulation into carriers (such as liposomes, micelles, and nanoparticles) to enhance accumulation at the desired site. Examples of second-generation photosensitizers are phthalocyanines, chlorins, and benzoporphyrins (Allison et al., 2004; Bazylińska et al., 2012). Figure 1.2 shows some examples of first- and second-generation photosensitizers.

Figure 1.2 Examples of first- and second-generation photosensitizers. 5′-Aminolevulinic acid is a precursor of protoporphyrin IX in the HEME biosynthesis pathway.

Second-generation photosensitizers are usually activated with wavelengths above 650 nm and have less phototoxicity, since their clearance from normal tissues is much faster than for porphyrins (Bazylińska et al., 2012). Chlorins are tetrapyrrole compounds derived from porphyrins, but with a reduced double bond in one pyrrole ring. This reduction red-shifts the absorption band to the far red (650–690 nm) and makes it stronger. This red-shifting is even more pronounced if a second double bond in a second pyrrole ring is reduced, generating a bacteriochlorin, although some criticism still remains about the stability of those compounds upon storage and the occurrence of photobleaching.

Phthalocyanines often contain a coordinated central metal atom that can determine the photodynamic properties of the molecule, that is, the singlet oxygen generation yield and the lifespan of the photosensitizer in the triplet state (Tynga et al., 2013). The four additional phenyl groups on these synthetic dyes cause solubility problems, which lead to the synthesis of phthalocyanines with peripherally attached sulfonic acid groups or with cationic groups to enhance water solubility (Fingar et al., 1993).

The US Food and Drug Administration has already approved a variety of photosensitizers for many biomedical applications, and many others have been tested in various clinical trials. Some examples are palladium-bacteriopheophorbide (TOOKAD) (Weersink et al., 2005), meta-tetrahydroxyphenylchlorin (Foscan®, Temoporfin) (Hopper et al., 2004), tin-ethyletiopurpurin (SnET2, Purlytin), Visudyne® (verteporfin, benzoporphyrin derivative monoacid ring A, BPD-MA; Novartis Pharmaceuticals), NPe6 (mono-Laspartyl chlorin e6, taporfin sodium, talaporfin, LS11; Light Science Corporation), Levulan® (5-aminolevulinic acid, a precursor of protoporphyrin IX) (Krammer and Plaetzer, 2008), and phthalocyanines (Pc4) (Allen et al., 2001).

Protocols for endobronchial and endoesophageal treatment (Dougherty, 2002), treatment for premalignant and early malignant diseases of the skin (actinic keratoses), bladder, breast, stomach, and oral cavity treatments (Dolmans et al., 2003) using PDT were also approved. Aminolevulinic acid (ALA) and its esters are examples of approved compounds for PDT. ALA is a precursor of protoporphyrin IX (PpIX) in the HEME biosynthetic pathway. ALA dehydratase combines two ALA molecules to form porphobilinogen, which is combined with three other porphobilinogen molecules by porphobilinogen deaminase. The new tetrapyrrole is enzymatically closed to form PpIX. The last step of the HEME pathway is the insertion of iron in the porphyrin by the enzyme ferrochelatase, which has a slower activity compared to the enzymes from the previous steps that leads to PpIX accumulation in the tissue (Tsai et al., 2004; Čunderlíková et al., 2011; Feuerstein et al., 2011).

1.3 Photochemistry of PDT

Once excited to a triplet state, the photosensitizer can generate reactive species through two broad kinds of reactions. In the type I reaction, the excited photosensitizer can gain an electron from a nearby cellular reducing agent such as nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH), becoming a radical anion. The triplet state photosensitizer can also react with another triplet state photosensitizer with an intermolecular electron transfer, producing a pair of cation and anion radicals. The latter is the radical which reacts with molecular oxygen with electron transfer to generate superoxide anion. Moreover, the triplet state photosensitizer can directly transfer an electron to molecular oxygen, generating a radical cation photosensitizer radical (which can be regenerated for further reactions if one electron is donated by a reducing agent) and superoxide anion.

In the type II reaction, the energy of the triplet photosensitizer is directly transferred to molecular oxygen (a molecule which is a triplet in its ground state), without electron transfer, to generate the excited-state singlet oxygen. Photosensitizers can undergo type I and II reactions at the same time, but the ration between those two reactions depends on the photosensitizer used and the microenvironment characteristics.

If the singlet oxygen generated reacts with the photosensitizer itself, the so-called photobleaching process occurs, in which the photosensitizer is inactivated and is no longer able to generate toxic compounds (Gomer, 1991; Boyle and Dolphin, 1996).

Superoxide radical is the main compound generated through a type I reaction, but its direct ROS effects are limited. If another electron is added to superoxide, it becomes hydrogen peroxide (a process called dismutation), and with addition of one more electron it generates hydroxyl radicals, which are highly reactive and thus can oxidize a great variety of biomolecules. When the oxidized biomolecules are lipids, hydroperoxyl radicals can be formed that can oxidize other biomolecules in a lipid peroxidation chain reaction, propagating the oxidative damage. Hydroxyl radicals can also be produced through the Fenton reaction (the donation of an electron from superoxide anion to ferrous or ferric iron, so that this metal can act as a catalyst to convert hydrogen peroxide into hydroxyl radical) (Halliwell and Gutteridge, 2006).

The lowest unoccupied molecular orbital (LUMO) of fullerenes can accept up to six electrons, making these compounds excellent Lewis acids, especially in the activated triplet state (Arbogast et al., 1992; Guldi and Prato, 2000; Koeppe and Saricift, 2006).

Fullerenes are more prone to produce oxidative species through a type I mechanism (generating superoxide, hydroxyl radical, and hydroperoxides) in polar solvents, compared to organic solvents where the singlet oxygen (type II) generation by fullerenes occurs more efficiently (Arbogast et al., 1991; Foote, 1994; Yamakoshi et al., 2003). When irradiated with visible light, fullerenes are excited from the ground singlet state (S0) to a short-lived excited singlet state (S1), which rapidly decays to a lower triplet state (T1) that has a lifetime between 50 and 100 µs (a spin-orbit coupling is necessary for this decay to occur, since it consists of a spin-forbidden intersystem crossing). When molecular oxygen is present, the triplet state fullerene is quenched rapidly (the lifetime is drastically reduced to about 300 ns), exciting the ground triplet oxygen to an activated singlet state oxygen (¹O2). If illuminated with light at 532 nm, C60 can generate singlet oxygen with a quantum yield near to 1.0 (the theoretical maximum) by energy transfer, at a rate of 2×10⁹ M−1 s−1 (Arbogast et al., 1991).

There is evidence that fullerenes such as C60 can act as antioxidants, a fact that seems contradictory to the role of fullerenes as photoinduced ROS generators. Some authors describe a scavenging action of fullerenes in the absence of light, theorizing that the double bonds could react with reactive species to form covalent bonds. This would prevent the reactive species from oxidizing other biomolecules. This hypothesis would have made it difficult to prove the photodynamic properties of fullerenes, until Andrievsky et al. solved the contradiction in 2009. The authors demonstrated that the antioxidant capacity of fullerenes was not a direct one, through covalent bond formation with ROS, but rather by the action of so-called ordered water (a coat of water associated with fullerene nanoparticles). According to them, these water molecules would slow down and even trap hydroxyl radicals for some time, enough for two radicals to react with each other, producing hydrogen peroxide, which is much less reactive (Gharbi et al., 2005; Cai et al., 2008; Lens et al., 2008; Spohn et al., 2009).

1.4 Fullerenes Acting as Photosensitizers

Since molecules with conjugated systems are more prone to act as photosensitizers and fluorophores, there is growing interest in the application of fullerenes as PDT mediators.

The first described fullerene was the C60, known at the time as Buckminsterfullerene or Buckyball. It was described by Culotta and Koshland in a 1991 editorial in Science (Culotta and Koshland, 1991). One problem that had to be overcome in order for fullerenes to be applied in the biomedical field was the significant hydrophobicity of fullerenes. A great variety of fullerenes have since been developed as functionalized structures with hydrophilic or amphiphilic properties, and those novel molecules have been shown to have high efficiency in PDT (Mroz et al., 2007).

One of the first described applications of fullerenes in the biomedical field was to produce DNA cleavage after illumination. Basically, supercoiled pBR322 DNA was incubated with a fullerene carboxylic acid under illumination by visible light, and the cleavage was only observed during the irradiation but not in the dark, with a considerable selectivity for guanine sites and an enhanced effect in an environment rich in deuterium oxide (D2O), where the lifetime of singlet oxygen is longer (Tokuyama et al., 1993). The effect of deuterium oxide was not observed in another study using fullerenes conjugated to a 285-base oligodeoxynucleotide, suggesting that the type I reaction was taking place in that case (An et al., 1996). In fact, water-soluble modified fullerenes showed evidence of singlet oxygen production (Liu et al., 2005), while the hydrophobic compounds tend to give more ROS generation through a type I mechanism.

Fullerenes present high efficiency for superoxide radical (type I reaction) and singlet oxygen generation (type II reaction) (Martin and Logsdon, 1987), and better photostability when compared to alternative photosensitizers such as tetrapyrroles, because the carbon backbone of fullerenes is less reactive to singlet oxygen than the tetrapyrrole structure. Therefore, fullerenes can generate reactive species longer than other photosensitizers. In spite of all these advantages, fullerenes have some disadvantages that could prevent their use in PDT, such as strong absorption in the blue and green regions of the spectrum, which leads to shallow light penetration. The covalent attachment of red-light-absorptive molecular antennas helps to overcome this disadvantage, since they absorb red light with high efficiency, and are able to transfer the energy absorbed to the fullerene cage, exciting it as if the buckyball was itself activated by red light (El-Khouly et al., 2006; Chiang et al., 2010). Another strategy is the application of femtosecond pulsed lasers at twice the one-photon photosensitizer excitation wavelength, which can lead to a two-photon absorption effect, although the small focused spot size needed to get the requisite peak power makes it necessary to scan the surface of the tissue to be treated.

1.5 Biocompatibility of Fullerenes

Although other molecules with polycyclic aromatic carbon rings, that is, benzene and anthracene, are usually thought to be carcinogenic, C60-derivatives are too big to intercalate with the DNA, compared to other less small aromatic compounds. The biodegradability was another concern about the use of fullerenes for biological applications, since non-degradable nanostructures tend to accumulate in the body or in the environment after use. Pristine C60 has been demonstrated to be nontoxic, but its insolubility in aqueous media prevents the use of this compound in vivo. This drawback can be overcome, however, if some drug-delivery strategies are applied to pristine and hydrophobic fullerenes, such as micelles (Akiyama et al., 2008; Kojima et al., 2008) or liposomal encapsulation (Yan et al., 2007; Akiyama et al., 2008), attachment of dendrimers (Hooper et al., 2008; Pan et al., 2009), or PEGylation (Tabata et al., 1997; Liu et al., 2007; Nitta et al., 2008). Cationic groups can be attached to fullerenes in order to enhance their biodistribution and selectivity (Yurovskaya and Trushkov, 2002; Nakamura and Isobe, 2003) and can enhance their binding to anionic groups present on cancer and bacterial cell surfaces. The most common ways to attach cationic groups to the carbon cage of fullerenes are cyclopropanation (Maggini et al., 1993; Brettreich et al., 2000; Wang et al., 2012; Sperandio et al., 2013) and pyrrolidination (Tegos et al., 2005; Thota et al., 2012; Wang et al., 2013). The design of new fullerene derivatives is described in more detail in the next section.

Fullerenes have been proven to be nontoxic for biological applications, after several toxicity studies were performed and reviews were published (Zakharenko et al., 1997; Chen et al., 1998; Ungurenasu and Airinei, 2000; Tagmatarchis and Shinohara, 2001; Bosi et al., 2003; Nakamura and Isobe, 2003; Fiorito et al., 2006; Satoh and Takayanagi, 2006).

1.6 Chemical Design of Fullerene Derivatives

Nanostructures such as fullerenes tend to accumulate in the body and in the environment, therefore their design should take into account the need for biodegradability and low levels of toxicity (Gharbi et al., 2005). Even though pristine C60 has been found to be nontoxic, its biomedical applications are not relevant due to the insolubility and tendency to aggregate in biological fluids. Several approaches can be applied to synthesize functionalized fullerene derivatives for in vivo applications, such as encapsulation into liposomes or micelles, dendrimer attachment, PEGylation, introduction into self-nanoemulsifying systems, encapsulation in cyclodextrins, attachment of antennae, and other chemical functionalization and drug-delivery strategies (Filippone et al., 2002; Yan et al., 2007; Akiyama et al., 2008; Doi et al., 2008; Hooper et al., 2008; Pan et al., 2009; Kojima et al., 2008; Zhao et al., 2008; Amani et al., 2010; Bali et al., 2010; Kato et al., 2010; Shakeel and Faisal, 2010). It is known that the covalent attachment of cationic groups is more useful than anionic ones for most biological applications, since cationic functional groups can increase the solubility as well as the chances to bind to anionic residues (via charge interactions) that are usually present on cancer cells and bacterial cell walls.

Among several chemical functionalization techniques that have been evaluated in previous studies (Yurovskaya and Trushkov, 2002; Nakamura and Isobe, 2003), cyclopropanation is one of the most suitable and commonly used (Maggini et al., 1993). Examples of cationic fullerene derivatives that have been synthesized through cyclopropanation are the compound LC14, or C60[>M(C3N6+C3)2]–(I−)10 (Wang et al., 2012; Sperandio et al., 2013) and the compound LC17 C70[>M(C3N6+C3)2]–(I−)10 (Brettreich et al., 2000). The structures of both compounds can be seen in Figure 1.3.

Figure 1.3 Chemical structure of (a) LC14 and (b) LC17.

To enhance water solubility and to increase the surface binding to bacteria cell walls (especially with –D-Ala-D-Ala– residues), decacationic functional moieties have been attached to C60, C70, and C84O2 fullerenes by incorporating multiple hydrogen-bonding interactions and positively charged quaternary ammonium groups to bind to anionic lipopolysaccharides and lipoteichoic acids (Wang et al., 2013). The structure includes two esters and two amide groups to give enough carbonyl and –NH groups within 20 Å that are necessary to provide multibinding sites in the presence of the pentacationic moiety C3N6+–OH at each side of the arm. A similar reaction can be performed with a malonate precursor arm M(C3N6+C3) in the preparation of the compounds LC18, LC19, and LC20, shown in Figures 1.4–1.6, respectively (Sperandio et al., 2013).

Figure 1.4 Chemical structure of the compound LC18.

Figure 1.5 Chemical structure of the compound LC19.

Figure 1.6 Chemical structure of the compound LC20.

Interesting results have been obtained with attachment of light-harvesting antennae onto the fullerene molecules. These chromophore structures, i.e., porphyrins, enhance the absorption of light at visible wavelengths by fullerenes once they are covalently bound. The hybrid molecule is also more efficient in generating singlet oxygen and in penetrating cells compared to the fullerene alone (Constantin et al., 2010).

Besides porphyrins, other molecular antennae have been tested with good success when attached to fullerenes. For instance, DPAF-C2M (dialkyldiphenylaminofluorene) can be covalently attached to C60 in order to facilitate the ultrafast energy and electron transfer in an intramolecular fashion, enhancing the PDT efficacy (Chiang et al., 2010). This compound absorbs at 400 nm, but also has a good two-photon absorption cross-section in the near-infrared region of the light spectrum. To red-shift the absorption of C60(>DPAF-C2M), some modifications can be performed in the molecule, such as a chemical conversion of the keto group to a 1,1-dicyanoethylenyl group, which is a stronger electron-withdrawing substituent. This modification shifts the absorption to between 450 and 550 nm and moreover causes an increased electronic polarization to the hybrid molecule. Furthermore, the modified molecule has a significant absorption of light in the red region (beyond 600 nm) and, consequently, a higher responsivity to longer tissue-penetrating wavelengths compared to C60(>DPAF-C2M).

According to El-Khouly and co-authors (2006), most of the HOMO electrons were delocalized over the DPAF-Cn part, while the LUMO electron density is located on the C60 cage. This fact leads to the conclusion that some charge-separated states might be generated by intramolecular electron transfer between the antennae (the electron donor) and the C60 cage (the electron acceptor) during the irradiation process. This is important to the generation of ROS such as superoxide and hydroxyl radicals, especially in polar solvents. In nonpolar solvents, the energy-transfer process that excites the ground molecule to the activated triplet state dominates and leads to a sixfold higher singlet oxygen generation.

1.6.1 Examples of the Synthesis of Mono- and Polycationic Fullerene Derivatives

The importance of cationic functional groups for the binding of fullerenes to anionic residues present on the bacteria cell wall was already mentioned earlier. The attachment of mono- or polycationic functionalities onto the fullerene cage is considered the best choice to increase the interactions with bacteria and to provide a better targeting of the fullerene to those microorganisms (Tegos et al., 2005). In this regard, multiple methods for chemical functionalization of fullerenes with cationic groups are available in the literature (Diederich and Gómez-López, 1999; Kadish and Ruoff, 2000; Yurovskaya and Trushkov, 2002; Hu et al., 2007) but, overall, the most suitable methods consist of the cyclopropanation or the pyrrolidination reactions, because of higher reproducibility and high consistency of the syntheses.

Studies by Lu et al. (2010) and by Mizuno et al. (2011) described fullerenes that were prepared by pyrrolidination reactions. The authors prepared a quaternized dimethylpyrrolidinium[60] fullerenyl monoadduct (BF4) and a trisadduct (BF6). C60 was treated with either 1.0 or 3.0 equivalents of sarcosine (N-methylglycine) and paraformaldehyde in toluene at the reflux temperature to generate the corresponding mono-N-methylpyrrolidino[60] fullerene (BF4) or several regioisomers of tris(N-methylpyrrolidino)[60] fullerene (BF6). Using methyl iodide the quaternization of those intermediates generated the corresponding monocationic and tricationic products BF4 and BF6 (Figure 1.7).

Figure 1.7 Chemical structure of the compounds (a) BL4 and (b) BL5.

When 1.0 or 2.0 equivalents of azomethine ylide (obtained by reacting piperazine-2-carboxylic acid dihydrochloride dissolved in methanol and trimethylamine in the presence of 4-pyridinecarboxaldehyde at the reflux temperature) were used in the reaction with C60 in toluene, the reaction product was the corresponding mono-piperazinopyrrolidino[60] fullerene or several regioisomers of bis(piperazinopyrrolidino)[60] fullerene derivatives. Again, the quaternization of these intermediates with methyl iodide led to the corresponding monocationic and dicationic products, this time BF22 and BF24, respectively.

Regarding cyclopropanation reactions as a means to functionalize C60, Wang and collaborators (2012) described recently the covalent attachment of a highly complex decacationic addend onto the fullerene molecule, and this method led to the generation of the products C60[>M(C3N6+C3)2] and C70[>M(C3N6+C3)2]. In order to produce enough effective multibinding sites to the bacterial cell wall, a sufficient number of –NH and carbonyl groups in a short length of around 20 Å and with a well-defined water-soluble pentacationic moiety N6+C3 at each side of the arm could be utilized. It was found to be possible by using the malonate precursor arm that included two amide and two ester moieties. This N6+C3 precursor can be used as a synthon for the structural modification of fullerenes (and in the future other photosensitizer structures), and it is derived from the quaternization of N,N′,N,N,N,N-hexapropyl-hexa(aminoethyl)amine precursor N6C3.

Briefly describing the most suitable cyclopropanation reaction for the preparation of the compounds C60[>M(C3N6+C3)2] and C70[>M(C3N6+C3)2], it starts with the preparation of well-defined fullerene monoadducts, i.e., the di(tert-butyl)fullerenyl malonates C60[>M(t-Bu)2] or C70[>M(t-Bu)2], followed by the transesterification reaction with 4-hydroxy-[N,N′,N,N,N,N-hexapropyl-hexa(aminoethyl)] butanamide (C3N6C3–OH) (which is the tertiary amine-precursor arm moiety). This reaction is carried out using trifluoroacetic acid as the catalytic reagent in order to afford protonated quaternary ammonium trifluoroacetate salt C70[>M(C3N6+C3H)2]. To reach the final product C70[>M(C3N6+C3)2], a neutralization of trifluoroacetic acid by sodium carbonate was performed, followed by a quaternization by methyl iodide in order to give decacationic quaternary ammonium iodide salts. For the final product C60[>M(C3N6+C3)2], a similar procedure was applied. These procedures combined comprise the first example of a synthetic protocol to incorporate a high number of cationic groups that do not need to use multiple addend attachments in order to preserve the unique optical and photophysical characteristics of fullerene cages.

1.6.2 Synthesis of Hexa-Anionic Fullerene Derivatives

Since fullerenes are highly hydrophobic molecules, they need to be solubilized by methods such as dispersion in micellar forms with the application of surfactants. However, micelles are known not to be very stable in biological systems. Bhonsle and co-workers (1998) described a strategy to overcome this instability. The procedure consisted of making micelle structures out of surfactants covalently attached directly onto the fullerene molecules. The authors synthesized the hybrid hexa(sulfo-n-butyl)-C60(FC4S), which led to structurally stable molecular micelles in aqueous media.

The reaction required the hexa-anionic C60 (C60⁶−) compound so that six sulfo-n-butyl arms could be attached on C60 in a one-pot reaction. The self-assembled structures were characterized by small angle neutron scattering (SANS) in deuterized water and by small angle X-ray scattering in water, and the analysis showed the formation of nearly monodisperse spheroidal nanospheres, with the sphere radius of gyration Rg around 19 Å, where the major axis is around 29 Å and the minor axis 21 Å for the ellipsoid-like aggregates, or an estimated long sphere diameter around 60 Å for the aggregates (Jeng et al., 1999).

This radius of gyration reveals a significant hydrophobic interaction between the core fullerene molecules that overcome the loose charge repulsion present at the surface of the micellar structures, and was found to remain constant even if the concentration was raised from 0.35 to 26 mM in deionized water. Another advantage was the possibility to form nanospheres even at a low concentration, in spite of the steric hindrance and hydrophobicity arising from the six sulfo-n-butyl arms that surround C60. The mean number of FC4S molecules per nanosphere, as revealed by SANS analysis, was found to be 6.5±0.7, and this led to the conclusion that the nanocluster structure consists of an octahedron-shaped nanosphere with FC4S molecules located at the vertex (Yu et al., 2005).

1.6.3 Synthesis of Chromophore-Linked Fullerene Derivatives

As mentioned above, the optical absorption of fullerenes like C60 is strong in the UVA region of the spectrum, but weak in most wavelengths of the visible spectrum. This could be solved when a light-harvesting donor chromophore antenna is attached to the fullerene molecule. Chiang described an approach in which an antenna was attached very closely to the C60 cage (within a contact distance of 2.6–3.5 Å), thus facilitating the ultrafast intramolecular energy and electron transfer from the chromophore antenna to the fullerene core and enhancing PDT efficacy (Chiang et al., 2010).

The authors used a specific donor antenna, the compound dialkyldiphenylaminofluorene (DPAF-Cn), which was first introduced to enhance optical absorption of light at 400 nm. Later, DPAF-Cn was modified by replacing the keto group using a highly electron-withdrawing 1,1-dicyanoethylenyl (DCE) bridging group, resulting in a dark burgundy-red solution of C60(>CPAF-Cn) derivatives. The optical absorption in wavelengths from 450 to 550 nm by the ground state molecule was increased after this structural modification.

Another example is the preparation of the compound C60(>CPAF-C2M), performed by a Friedel–Crafts acylation of 9,9-dimethoxyethyl-2-diphenylaminofluorene with bromoacetyl bromide and in the presence of AlCl3, resulting in 7-bromoacetyl-9,9-dimethoxyethyl-2-diphenylaminofluorene. Then, a cyclopropanation reaction was performed with C60, resulting in the product C60(>DPAF-C2M). Finally, this product was treated with malononitrile and pyridine in the presence of titanium tetrachloride in dry toluene to yield the final product C60(>CPAF-C2M) after purification with chromatography (Figure 1.8).

Figure 1.8 Chemical structure of the compounds (a) C60(>CPAF-C2M) and (b) C60(>DPAF-C2M).

1.7 Photochemical and Photophysical Properties of Fullerenyl Molecular Micelles and Chromophore–Fullerene Conjugates

Fullerenes can act as photosensitizers. In other words, the excitation of C60 and other fullerene derivatives by light induces a singlet fullerenyl excited state, which is transformed to the correspondent triplet excited state via intersystem crossing. This conversion occurs with nearly quantitative efficiency (Guldi and Prato, 2000). The energy can then be transferred to molecular oxygen from the triplet fullerene derivatives in aerobic media, producing singlet molecular oxygen, which is highly reactive and has a short lifetime. This is not only the main process that provides the photodynamic efficiency of fullerenes, but it is the key photocatalytic effect behind the mechanism of PDT with most other photosensitizers.

This photocatalytic effect is partially impaired with the functionalization and molecular modifications that are performed in order to enhance solubility, optical absorption, and biocompatibility. A marked reduction in the singlet oxygen production quantum yield is observed in fullerene derivatives after functionalization. Examples were described before, including Bingel-type malonic acid, C60[C(COOH)2]n and malonic ester, C60[C(COOEt)2]n fullerene adducts, with an evident decrease in the singlet oxygen production levels proportional to the number of addends attached to the core fullerene molecule (Guldi and Asmus, 1997). Compared to C60, only 13% of singlet oxygen production quantum yield was observed when the number of adducts reaches six (the hexaadduct) (Guldi and Asmus, 1997).

Fullerenes in micellar forms tend to conserve their high singlet oxygen production ability. Bensasson and collaborators (2001) showed that molecular micellar compound FC4S can still produce high levels of singlet molecular oxygen through a mechanism that differs from the one observed for Bingel-type malonic hexaadducts of C60. The authors demonstrated this by directly detecting singlet oxygen emission at 1270 nm while the self-assembled FC4S nanospheres were irradiated with light at 500–600 nm.

The mechanisms of photodynamic activity showed by fullerenes conjugated with light-harvesting electron-donor chromophores diverge from those previously described. In this case, such conjugate systems (i.e., C60(>CPAF-Cn) derivatives) have their photophysical properties dependent on the photoexcitation of either the fullerene moiety (requiring irradiation with UVA wavelengths) or the DPAF-Cn moiety (requiring irradiation with light at visible wavelengths, mainly within 500 and 600 nm) (Chiang et al., 2010). In fact, since the visible light absorption by the DPAF-Cn moiety is considerably higher than the fullerene cage itself, the former group serves as a light antenna, and the formation of photoexcited ¹(DPAF)*-Cn moiety should be preferentially considered as the early event in the photodynamic mechanism of these fullerene derivatives.

If the keto group of C60(>DPAF-Cn) is changed to the 1,1-dicyanoethylenyl group of C60(>CPAF-Cn), the optical absorption of the conjugated molecule is markedly red-shifted. The photoexcitation of C60(>DPAF-Cn) and C60(>CPAF-Cn) allows the transfer of electrons from their HOMO, which is delocalized over the DPAF-Cn -Cn) is believed to be the most stable charge-separated state in polar solvents. Charge-separated states like this are believed to be generated by photoinduced intramolecular transfer of electrons between dyphenylaminofluorene (DPAF-Cn) donors and C60> acceptor moieties.

The fluorenyl fluorescence that is commonly observed in C60(>CPAF-Cn) and C60(>DPAF-Cn) monoadducts is significantly quenched by this process. This quenching is observed even during energy transfer events of C60(>CPAF-Cn) (which is favored in nonpolar solvents), since the fluorescence lifetime of the model compound ¹CPAF*-C9 (not higher than 241 ps) is markedly lower than the lifetime observed with the keto analog Br-¹DPAF*-C9 (2125 ps). This observation indicates that an easy photoinduced intramolecular charge polarization process occurs that forms the corresponding [C=C(CN)2]−•-DPAF*•-C9 charge-separated state. This process would facilitate the formation of C60−•(>CPAF+•-Cn) during the subsequent electron-transfer event.

1.8 Fullerenes for Antimicrobial Inactivation

The discovery of antibiotics in the middle of the twentieth century brought a new era to the control of infections (Maisch, 2009). However, the inappropriate and excessive use of these drugs led to the worldwide emergence and rapid spread of antibiotic resistance that we observe today. Some early discovered ways to deal with infections, that is, treatments using photoactivated agents, were forgotten during the age of antibiotics, but the antimicrobial PDT approach has emerged once again since the problem of antibiotic resistance has become a global concern, and has initiated a critical search for new antimicrobial methods, toward which bacteria will be unable to develop resistance (Talan, 2008). Therefore, in recent decades PDT has become a promising strategy to treat infections caused by multidrug-resistant microorganisms (Hamblin and Hasan, 2004). PDT acts against a broader spectrum of different classes of microorganisms compared to antibiotics, and does not cause the strains to become resistant (Hancock and Bell, 1988).

In order to be considered a good photosensitizer for antimicrobial PDT, the compound must achieve many criteria. It must be able to selectively inactivate multiple classes of microorganisms with low concentrations and low light doses, presenting low to zero levels of dark toxicity. The quantum yields of the triplet state and singlet oxygen must be high as well. Cationic fullerenes have been demonstrated to fulfill most of the above-mentioned desired criteria, killing a broad variety of bacteria when irradiated with light in the visible spectrum (400–700 nm), although most of the cationic fullerenes present a slightly higher level of dark cytotoxicity (Tegos et al., 2005). Our laboratory demonstrated the effect of some fullerene derivatives on light-mediated killing (more than 4 logs) of Staphylococcus aureus with drug doses as low as 1 µM and light doses of 1 or 2 J/cm². These findings agree with the data available in the literature regarding the comparable efficiency of cationic fullerenes compared to other photosensitizers (Merchat et al., 1996; Minnock et al., 1996; Demidova and Hamblin, 2004, 2005; Hamblin and Hasan, 2004).

Noncationic compounds, on the other hand, are less effective to photoinactivate Gram-negative strains such as Escherichia coli (Hamblin and Hasan, 2004). This can be explained by the structure of bacterial outer layers: while Gram-positive cells have relatively permeable outer layers composed of peptidoglycans, lipoteichoic acid, and beta-glucan molecules (which allows cationic and noncationic photosensitizers to reach the plasma membrane and generate oxidative damage to this structure upon illumination), Gram-negative cells have an outer membrane of lipopolysaccharides (LPS) that prevents this diffusion. Cationic compounds are able to displace divalent cations that are important for the attachment of LPS, weakening the structure of the outer layer and enhancing the permeability of photosensitizers (self-promoted uptake) (Hancock and Bell, 1988; Lambrechts et al., 2004).

Besides the interference with cell wall structure and impairment of membrane integrity, several mechanisms can explain fullerene antimicrobial activity, such as interference with nucleic acid and folate synthesis or impairment of ribosomal function. There is evidence indicating that type I reactive species, that is, superoxide and hydroxyl radicals, are more effective against Gram-negative microbial cells than is singlet oxygen (Dahl et al., 1989; Valduga et al., 1993). As mentioned above, water-soluble fullerene derivatives prioritize type I reactions and, therefore, are the most promising compounds for photodynamic inactivation of these microorganisms. Singlet oxygen, on the other hand, is more effective for Gram-positive bacteria inactivation, possibly because it diffuses more easily into the porous cell wall of Gram-positive bacteria, reaching deeper and more sensitive sites (Dahl et al., 1989; Valduga et al., 1993).

In fact, increasing the number of cationic groups and, consequently, decreasing the degree of hydrophobicity, results in enhanced photodynamic inactivation of both Gram-positive and Gram-negative bacteria due to the increased interaction with negatively charged structures of microbial membranes. Results from our laboratory indicate that, besides the difference between several polycationic fullerene derivatives, the number of carbon atoms in the fullerene cage influenced the activity of the compounds as well. Gram-positive bacteria were more susceptible to photodynamic damage when a cationic C60 derivative was used, compared to the cationic C70. Interestingly, the opposite effect was observed with Gram-negative bacteria.

Promising results regarding fullerene-mediated PDT activity and antimicrobial effects have already been published by several authors. A tricationic fullerene derivative (BF6), for instance, was found to be effective against both Gram-negative and Gram-positive bacteria, as well as yeasts in vitro (Tegos et al., 2005). Our lab tested the same compound in vivo, on a potentially lethal model of murine wound infected with Pseudomonas aeruginosa and Pseudomonas mirabilis (Gram-negative bacteria), and found that the PDT using fullerene was able to prevent death of mice with wounds infected by P. mirabilis, and in mice whose wounds were infected with P. aeruginosa when PDT was combined with a suboptimal dose of antibiotics. Therefore, fullerenes can be used to treat wounds infected with virulent Gram-negative bacteria alone, or they can be used with a low dose of antibiotics, synergistically controlling the infection and preventing bacterial regrowth (Lu et al., 2010).

Another promising result was achieved with photoactivated fullerenes on third-degree burns, which are particularly susceptible to bacterial infection due to the destruction of the skin barrier of dead cells and the diminished blood perfusion that takes place on the burned area (preventing the antibiotics from reaching the burned tissue) (Ollstein and McDonald, 1980). The gold standard procedure for third-degree burns is excision and skin grafting (Saffle, 2009), but there is still the problem of superimposed infections, especially if the infectious agent is a Gram-negative bacterium. It is known that Gram-negative bacteria tend to cause sepsis much more easily than Gram-positive bacteria. PDT offers valuable help in controlling pathogens that infect burned areas, and offers advantages over topical antimicrobials because they can be used for drug-resistant strains (Dai et al., 2010).

Our laboratory synthesized a C70-based compound similar to one previously available in the literature and which was used to treat Gram-negative infections in vitro. We used the new compound in vivo to treat infection on a third-degree burn with bioluminescent Gram-negative bacteria, and the results that the modified compound had an increased yield of hydroxyl radicals under UVA irradiation, possibly due to the attachment of an additional deca(tertiary-ethylenylamino) malonate arm to the C70 cage (generating the compound LC18) that acted as an effective electron donor (Huang et al., 2013). The phenomenon of intramolecular energy transfer from tertiary amine attachments to the fullerene cage in an aqueous environment upon short-wavelength photoinduction is already known, and LC18 was shown to be a promising hydroxyl radical generator due to this effect.

1.9 Conclusions

Fullerenes have been studied in recent years as potential photosensitizers that could mediate PDT of a wide number of diseases, among other applications. Most of the studies consisted of in vitro experiments, where viruses, microorganisms, or even cancer cells were incubated with fullerenes (or their derivatives) and irradiated with light under 400 nm. In this chapter, we pointed out how the unique optical properties and the unusual photochemical mechanism of fullerenes made these compounds suitable for use as photodynamic agents to destroy pathogenic microorganisms and to treat infections. Chemical modifications to improve biocompatibility, optical absorption, and solubility can generate fullerene derivatives that produce a substantial amount of ROS, such as superoxide anion in a type I photochemical reaction. This ROS production occurs in a process involving electron transfer from the excited triplet state fullerene to molecular oxygen in aqueous solution. Hydroxyl radicals, one of the most reactive and cytotoxic ROS, can also be generated from hydrogen peroxide in result of the photoinduced electron transfer