Functionalized Fullerenes in Photodynamic Therapy

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ReviewJournal of

Biomedical NanotechnologyVol. 10, 1918–1936, 2014

www.aspbs.com/jbn

Functionalized Fullerenes in Photodynamic Therapy

Ying-Ying Huang1�2, Sulbha K. Sharma3, Rui Yin1�2�4, Tanupriya Agrawal1�2,Long Y. Chiang5, and Michael R. Hamblin1�2�6�∗1Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA 02114, USA2Department of Dermatology, Harvard Medical School, Boston, MA 02115, USA3Raja Ramanna Centre for Advanced Technology, Indore, MP 452013, India4Department of Dermatology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China5Department of Chemistry, Institute of Nanoscience and Engineering Technology, University of Massachusetts, Lowell, MA 01854, USA6Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA

Since the discovery of C60 fullerene in 1985, scientists have been searching for biomedical applications of this mostfascinating of molecules. The unique photophysical and photochemical properties of C60 suggested that the moleculewould function well as a photosensitizer in photodynamic therapy (PDT). PDT uses the combination of non-toxic dyes andharmless visible light to produce reactive oxygen species that kill unwanted cells. However the extreme insolubility andhydrophobicity of pristine C60, mandated that the cage be functionalized with chemical groups that provided water solubilityand biological targeting ability. It has been found that cationic quaternary ammonium groups provide both these features,and this review covers work on the use of cationic fullerenes to mediate destruction of cancer cells and pathogenicmicroorganisms in vitro and describes the treatment of tumors and microbial infections in mouse models. The design,synthesis, and use of simple pyrrolidinium salts, more complex decacationic chains, and light-harvesting antennae thatcan be attached to C60, C70 and C84 cages are covered. In the case of bacterial wound infections mice can be savedfrom certain death by fullerene-mediated PDT.

KEYWORDS: Fullerene, Photodynamic Therapy, Reactive Oxygen Species, Cancer, Infections, Electron Transfer, Singlet Oxygen,

Cationic Charge.

CONTENTSIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1918Traditional Photosensitizers . . . . . . . . . . . . . . . . . . . . . . . . . 1920

Photophysics and Photochemistry of PDT . . . . . . . . . . . . . . 1921Fullerenes as Photosensitizers . . . . . . . . . . . . . . . . . . . . . . . 1921

Photophysics and Photochemistry ofFullerenes and Derivatives . . . . . . . . . . . . . . . . . . . . . . . . 1921

Design of Fullerene Derivatives . . . . . . . . . . . . . . . . . . . . . . 1922Examples of the Synthesis of Monocationic andPolycationic Fullerene Derivative . . . . . . . . . . . . . . . . . . . 1924Synthesis of Hexa-Anionic Fullerene Derivatives . . . . . . . . . 1926Synthesis of Chromophore-Linked Fullerene Derivatives . . . . . 1926Photochemistry and Photophysics of Fullerenyl MolecularMicelles and Chromophore-Fullerene Conjugates . . . . . . . . . 1926

Anticancer Effect of Fullerene-PDT . . . . . . . . . . . . . . . . . . . . 1927In Vitro Anti-Cancer PDT with Fullerenes . . . . . . . . . . . . . . 1927In Vivo Photodynamic Therapy of Cancer . . . . . . . . . . . . . . 1928

Antimicrobial Effect of Fullerenes . . . . . . . . . . . . . . . . . . . . . 1929

∗Author to whom correspondence should be addressed.Email: [email protected]: 6 February 2014Accepted: 5 March 2014

Antimicrobial Effects In Vitro . . . . . . . . . . . . . . . . . . . . . 1930Antimicrobial Effect In Vivo . . . . . . . . . . . . . . . . . . . . . . 1931

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1933Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1933References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1933

INTRODUCTIONPhotodynamic therapy (PDT) is based on the administra-tion of nontoxic light absorbing dyes called photosensitiz-ers (PS), either systemically, locally or topically, followedby irradiation of harmless visible to near infrared (NIR)light, in the presents of oxygen, leading to the generationof cytotoxic reactive oxygen species (ROS).1 Dual selec-tivity of PDT can be obtained by the therapeutic gradientof photosensitizer concentration between tumor and nor-mal tissues and precise delivery of light exposure withinthe tumor.2 However, the major limitations of PDT arenon-specific cellular uptake of PS by normal cells andshort light penetation depth. PDT is a widely recognizedvaluable treatment option for neoplastic and non-malignant

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diseases. Recently with the development of fiber-optic sys-tems, light can be delivered accurately into many partsof the body for the treatment of tumors. Therefore, PDTapplications have been expended to many endoscopicallyaccessible tumors, including lung cancer, superficial gas-tric cancer, head and neck cancer, cervical cancer and blad-der cancer. In general, there are a number of advantages forPDT over chemotherapy and radiotherapy: PDT shows no

Ying-Ying Huang, M.D., is a researcher in Dr. Michael Hamblin’s lab in WellmanCenter for Photomedicine at Massachusetts General Hospital, an Instructor of Derma-tology at Harvard Medical School. She received her M.D. from China in 2004. Sheearned her M.Med in Dermatology in China and she was trained as a dermatologist.She has been at MGH Wellman Center for 5 years. Her research interests lie in theareas of photodynamic therapy (PDT) for infections, cancer and mechanism of lowlevel light therapy (LLLT) for traumatic brain injury. She has published 48 peer reviewarticles and 15 conference proceedings and book chapters. She is the co-editor of newlyreleased “Handbook of Photomedicine.”

Sulbha K. Sharma, Ph.D. is a visiting fellow at Raja Ramanna centre for advancedtechnology, Indore, India. Earlier she was a postdoctoral fellow at Dr. Hamblin’s lab atThe Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA.She completed her Ph.D. from Laser Biomedical section and instrumentation divisionat Raja Ramanna centre for advanced technology, Indore, India. She has published25 peer-reviewed articles 7 conference proceedings and 4 book chapters. Her researchinterests are anticancer photodynamic therapy and low level light therapy.

Rui Yin, M.D., Ph.D. is an Associate Professor of Dermatology, Southwest Hospi-tal, Third Military Medical University, and a visiting associate professor of WellmanCenter for Photomedicine at Massachusetts General Hospital. Her research interests liein the areas of photodynamic therapy for infections and cancer, the electron transfermechanisms of photodynamic reaction. She has published 17 peer-reviewed articlesin English, over 40 peer-reviewed articles in Chinese, over 30 conference proceeding,2 book chapters. She is a reviewer for 7 journals and serves on National Natural ScienceFoundation of China as a grant reviewer. She is also a committee member of China Der-matologist Association and China Medical Association. In 2011, Dr. Yin was honoredas one of Top 10 National Outstanding Young Dermatologist by China DermatologistAssociation.

Tanupriya Agrawal, M.D., Ph.D. is a postdoctoral fellow at Dr. Hamblin’s lab atThe Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA.Prior to this, she finished medical school at N.S.C.B Medical College, Jabalpur, Indiafollowed by Ph.D. in Biomedical Sciences at Creighton University, Omaha, NE. Shehas completed United States Medical Licensing Examination (USMLE) and certified byEducational Commission for Foreign Medical Graduates (ECFMG). She has published6 peer-reviewed articles and 12 conference proceedings. She is investigating the roleand underlying molecular mechanisms of low level laser therapy in neurogenesis andsynaptogenesis in mouse model of traumatic brain injury.

long-term side effects when an effective PS is employed;it is a minimally-invasive procedure with little or no scar-ring after the site heals as compared to surgery; it candeliver highly targeted precision at the disease site; therecan be repeatable treatments at the same site if needed;PDT can be less costly than other cancer treatments; it maytake only a short period of time for each session allowingtreatment as an outpatient. The main limitation of PDT

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Long Y. Chiang, Ph.D. is a Professor at the Department of Chemistry, University ofMassachusetts Lowell. His research interests lie in the areas of design and synthesis ofultrafast broadband photoresponsive linear and nonlinear multiphoton energy absorptivefullerenyl nanostructures and polycationic, nano-PDT drugs in combination of photoen-ergy, in one-photon absorptive 1PA-PDT and NIR two-photon absorptive 2PA-PDT,to kill multiantibiotic-resistant bacteria and cancer/tumor cells. He has published 269peer-reviewed articles, book chapters, review articles, and awarded 39 patents. He was achairman of several MRS symposia and a member of international advisory committeeof ICSM. He was a member of editorial board and regional editor of two journals andis for one journal.

Michael R. Hamblin, Ph.D. is a Principal Investigator at the Wellman Center for Pho-tomedicine at Massachusetts General Hospital, an Associate Professor of Dermatologyat Harvard Medical School and is a member of the affiliated faculty of the Harvard-MITDivision of Health Science and Technology. His research interests lie in the areas ofphotodynamic therapy (PDT) for infections and cancer, and in low-level light therapy(LLLT) for wound healing, arthritis, traumatic brain injury and hair regrowth. He haspublished 250 peer-reviewed articles, over 150 conference proceedings, book chapters,has edited 3 major textbooks, and holds 8 patents. He is Associate Editor for 7 journalsand serves on NIH Study Sections. In 2011 Dr Hamblin was honored by election as aFellow of SPIE.

is its action occurs only at areas where light can reach.This implies that the major sites of tumors treated by PDTare the lining of organs or just under the skin that can bereached by the light source.

TRADITIONAL PHOTOSENSITIZERSPhotosensitizers can be categorized by their chemi-cal structures as porphyrins (tertrapyrroles) and non-porphyrins.3 First, second or third generation PS are termsfurther used to label tetrapyrrole-derived PS. Porphyrinsare derived from the tetrapyrrole aromatic macrocyclewhich is a major component of many naturally occuringpigments such as heme, chlorophyll and bacteriochloro-phyll. Porphyrins contain a structure with a 22 �-electronsystem which gives rise to their long wavelenth absorptionof light.4 Hematoporphyrin (Hp), hematoporphyrin deviva-tive (HpD) and Photofrin are referred to as first generationPS. HpD and Photofrin have been widely used in clinicalfor cancer.5 However, the side effects associated with 1stgeneration PS, such as prolonged skin photosensitiztionand suboptimal tissue penetation of the 630 nm lighthave stimulated interst in development of new PS. Secondgeneration of PS are chemically purified or synthetictetrapyrrole derivatives, which absorb longer wavelengthlight and cause less skin photosensitization. Some promis-ing second generation photosensitizers have been approvedor tested in clinical trials.6 These include, but are notlimited to, palladium-bacteriopheophorbide (TOOKAD),7

meso-tetra-hydroxyphenylchlorin (Foscan®, Temoporfin),8

tin-ethyletiopurpurin (SnET2, Purlytin), Visudyne®

(verteporfin, benzoporphyrin derivative monoacid ring A,BPD-MA; Novartis Pharmaceuticals), NPe6 (mono-L-aspartyl chlorin e6, taporfin sodium, talaporfin, LS11;Light Science Corporation), Levulan® (5-aminolevulinicacid, a precursor of protoporphyrin IX),9 and phthalocya-nines (Pc4).10 The term “third generation PS” refers tothe 2nd generation PS bound to carriers such as antibodyconjugates11 liposomes and other targeted structures forincreasing the selectively for tumor tissues. Although themajority of PS are porphyrin derivatives, non-porphyrinPS are being employed to improve PDT efficacy andminimal side effects. Synthetic, non-naturally-ocurring,conjugated or expanded pyrrolic ring systems are anotherclass of non-porphyrin PS, including texaphyrins, por-phycenes, phthalocyanines and naphthalocyanines. Othercompounds that have been studied as PS are not derivedfrom the tetrapyrrole backbone, but can be classedas miscellaneous dyes including chalcogenopyryliumdyes,12 phenothiazinium dyes including methylene blueand toluidine blue,13 Nile blue derivatives,14 hydroxy-lated perylenequinones such as hypericin,15 BODIPYderivatives,16 squaraines17 etc. These compounds haveprovided a new focus to the field of PDT.It is generally accepted that the characteristics that the

ideal PS used against cancer should possess are:(1) single compound with known composition and goodstability,(2) preferential uptake and retain in the target tumourtissue,(3) minimal toxicity in the absence of light to preventharmful side-effect to the surrounding normal tissue,

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(4) high quantum yield of triplet state and ROS generationand(5) high molar extinction coefficient to minimize the doseof PS needed to achieve the desired PDT effect,(6) intrinsic fluorescence to permit their detection by opti-cal imaging (microscopy) techniques, and(7) high absorbance, particularly in the red part of thespectrum, which leads to a deeper light penetration into thetissues; for instance, the light depth penetration at 500 nmis about 4 mm, whereas at the 600–800 nm range it isabout 8 mm2.

Photophysics and Photochemistry of PDTMost PS have 2 electrons with opposite spins located inan energetically lower energy orbital, the so-called high-est occupied molecular orbital (HOMO), in their ground(usually single) state. Absorption of light (photons) leadsto an excited singlet state by the elevation of one elec-tron with unchanged spin to a higher energy obital,called the lowest unoccupied molecular obital (LUMO).18

This excited singlet PS is short-lived (nanoseconds) andemits excess energy as fluorescence and/or heat. Alterna-tively, an excited PS may undergo an intersystem crossing(ISC) by inverting spin of one electron to form a rel-atively long-lived (microseconds to milliseconds) tripletstate.The excited triplet PS can either decay radiationlessly to

the ground state or can survive long enough to transfer itsenergy to molecular oxygen O2 (ground triplet state) andproduce excited state singlet oxygen (1O2). This reactionis referred to as a Type II process. A Type I process canalso occur whereby the PS reacts directly with neighbor-ing molecules and gains or donates an electron to forma radical. Subsequent electron donation from the PS radi-cal anion to oxygen produces the superoxide anion radical(O−•

2 ). Therefore, the efficient ISC process giving a highquantum yield of triplet state is essential for the generationof ROS. Strong absorbtion of light, high triplet state quan-tum yield (effective ISC) and a long-lived triplet excitedstate are required to be an ideal PS. However, there hasnot yet been established a clear relationship between theefficiency of ISC and the chemical structure.

FULLERENES AS PHOTOSENSITIZERSBesides occupying an important place in biomedicine,nanoparticles have also been shown to have potential toact as a photosensitizing drug in PDT. Fullerenes are con-sidered to have advantages as potential PS on the basisof certain favorable PDT characteristics when comparedto conventional PS with the tetrapyrrole structure.19 Thefullerenes are known for their photostability, a prerequi-site to behave as an effective PS, and they undergo rel-atively less photobleaching than the tetrapyrroles. Theycan also be modified to get the desired the degreelipophilicity by chemical functionalization.20 There is

the option of attaching the light-harvesting antennae tothe fullerenes to increase the quantum yield of produc-tion of ROS. Fullerenes can self-assemble into vesiclescalled fullerosomes that can act as multivalent drug deliv-ery vehicles with the possibility of different targetingproperties.21

Fullerenes have been said to offer “a wide open playingfield to chemists”22 by providing synthetic opportunities toattach a wide variety of hydrophilic or amphiphilic sidechains or fused-ring structures to the spherical C60 core.22

Furthermore, fullerenes have hollow interiors, where otheratoms, ions or small clusters can be entrapped and formendohedral fullerenes. Those fullerenes that encapsulatemetal atoms are called endohedral metallofullerenes.23

Hydroxylation (attaching OH-groups) is the most com-mon functionalization, which renders the molecule morehydrophilic,24 but other polar adducts will also have thesame effect.25

There are also several unfavorable characteristics offullerenes for PDT, but by applying different strategiesthey can be overcome. The main absorption of fullerenesoccurs in the UV, blue and green regions of the spectrumwhile the absorption of tetrapyrrole PS (such as chlorins,bacteriochlorins, and phthalocyanines) shows substantialpeaks in the red or far-red regions where the penetra-tion of light into tissue is much deeper. This majordisadvantage of fullerenes however can be overcome inseveral ways, for example by chemical attachment of oneor more red-wavelength absorbing antennae onto a C60

cage.26 The absorption of many light photons simulta-neously can be achieved by the attachment of multiplelight-harvesting antennae on one C60 cage. The unfavor-able absorption spectrum of fullerenes can also be over-come by using optical clearing agents to the tissue27–30 orby using two-photon excitation where two NIR photonsare simultaneously delivered in a very short pulse to beequivalent to one photon of twice the energy and shorterwavelength.31–35

Photophysics and Photochemistry ofFullerenes and DerivativesFullerenes with attached side chains, called “functional-ized fullerenes,” are known to demonstrate high efficiencyin the formation of singlet oxygen, hydroxyl radicals andsuperoxide anions, which are considered as effective medi-ators of PDT. The photophysics of fullerenes is quitefavorable for PDT. The absorption spectra of a typicalset of mono-substituted, bis-substituted and tris-substitutedfullerenes show almost monotonic decay between 300 and700 nm.20 When C60 is irradiated with visible light, it isexcited from the S0 ground state to a short-lived (< 1.3 ns)S1 excited state. The S1 state quickly decays to tripletstate. The triplet yield is 1 and the lifetime is as longas 50–100 �s. By energy transfer mechanism (Type II)there is the generation of singlet oxygen (1O2) by quench-ing of fullerenyl T1 state in the presence of dissolved

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Figure 1. Schematic of PDT mediated by fullerenes.

molecular oxygen. At 532 nm excitation the singlet oxy-gen quantum yield (��) for this process is close to theo-retical maximum, 1.0.36 Calculations in 1986 by Haddonet al. had indicated that the lowest unoccupied molecularorbital (LUMO) of C60 would be low-lying, triply degen-erate, and, hence, capable of accepting up to 6 electrons.37

The pristine C60 itself alone is not a good PS due to thevery weak absorption of visible light and water insolu-bility. However, C60 is an ideal spin converter due to itsefficient inherent ISC and a low S1 state energy level(about 1.72 eV). When a visible-light-harvesting antennais attached onto C60 to produce C60-dyads, it turns out tobe a potentially ideal PS.38 Figure 1 shows a schematicoutline centered upon a Jablonski diagram of PDT medi-ated by fullerene and its derivatives.Both pristine and functionalized fullerenes have the

potential to produce ROS after illumination.39 The ROSproduced by fullerene derivatives during illumination areinclined towards Type I photochemical products (super-oxide radical, hydroxyl radical, lipid hydroperoxides, andhydrogen peroxide), compared to the standard Type II ROS(singlet oxygen) for most PS. During this process they areable accept electrons very efficiently as many as six toeach C60 cage.40�41 It is thought that the reduced fullerenetriplet or radical anion can transfer an electron to molec-ular oxygen, forming the superoxide anion radical.42 Oneapparent incongruity that arises in this area needs to beaddressed. It is a well-known fact that fullerenes can actas antioxidants, and that C60 and derivatives can act asscavengers of ROS in the absence of light. One clue thatmight explain this inconsistency was proposed in 2009, byAndrievsky et al.43 who showed that the major mechanismby which hydrated C60 can inactivate the highly reactiveROS, hydroxyl radical, not by covalently scavenging theradicals but rather by action of the coat of “ordered water”that was linked with the fullerene nanoparticle.44 One ofthe explanations is that hydroxyl radicals can be slowed

down or trapped for a sufficient time allowing the tworadicals to react with each other, which produce the com-paratively less-reactive ROS, hydrogen peroxide. However,the mechanism may be significantly different with morewater-compatible C60 derivatives.45

DESIGN OF FULLERENE DERIVATIVESOne of the concerns that have been raised about the useof fullerenes concerns their biodegradability, as nano-structures have the possibility of accumulation in the bodyduring blood circulation or in the environment after use.46

Though it has been shown that pristine C60 is nontoxicits insolubility and its pronounced tendency to aggre-gate decreases its potential to be useful in biomedicine.Many strategies have been demonstrated or applied toeither solubilize or modify fullerenes for improvingtheir utility in drug-delivery and medical applications.The following approaches: liposomes;47–49 micelles;50�51

dendrimers;52�53 PEGylation;53–56 self-nanoemulsifyingsystems (SNES);57–60 encapsulation in cyclodextrins.47�61�62

have all been explored by various groups throughout theworld to overcome this problem with fullerenes.It is known that cationic functional groups provide

good solubility for molecules of diverse structures andalso have the potential to bind to the anionic residuespresent on cancer cells and also on the bacterial cell wallvia static charge interactions. For these reasons cationicgroups can be considered a good choice for attachmenton the fullerene cage. A number of chemical function-alization techniques for derivatizing fullerenes have beenevaluated.63�64 Among them, general suitable methods forthe preparation of cationic fullerene derivatives includecyclopropanation65 e.g., C60[>M(C3N

+6 C3)2] − (I−)10

(LC14, Fig. 2(a))66�67 and C70[>M(C3N+6 C3)2]− (I−)10

(LC17, Fig. 2(b))68 and pyrrolidination e.g., quaternizeddimethylpyrrolidinium fullerenyl monoadduct (BF4,20

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Figure 2. The chemical structure of fullerene derivatives applied in PDT studies.

Fig. 2(c)) and trisadduct (BF6,69 Fig. 2(d)) and structuressuch as LC22, C60[>DPAF(MN+

6 C3)2]−(I−)10, and LC24,C70[>M(C3N

+6 C3)2]2− (I−)20.70�71

The cyclopropanation reaction of C60 functionaliza-tion was recently applied to attach a highly complexdecacationic moiety to the fullerene cage leading tothe formation of fullerenes bearing ten positive charges.The decacationic functional moieties of C60, C70, andC84O2 fullerenes were designed to increase both thewater-solubility and provide surface binding interactionswith –D-Ala-D-Ala residues of the bacteria cell wall byincorporating multiple H-bonding interactions and pos-itive quaternary ammonium charge to bind to anioniclipopolysaccharides and lipoteichoic acids.70 The struc-ture included two esters and two amide moieties to givea sufficient number of carbonyl and –NH groups in ashort length of ∼ 20 Å to provide effective multi-bindingsites with the presence of a well-defined water-solublepentacationic moiety C3N

+6 C3-OH at each side of the

arm. A similar reaction sequence with a malonate pre-cursor arm M(C3N6C3)2 was also employed in the prepa-ration of C70[>M(C3N

+6 C3)2][>M(C3N6C3)2] − (I−)10

(LC18, Fig. 2(e)), C84O2[>M(C3N+6 C3)2]− (I↓)10 (LC19,

Fig. 2(f)), C84O2[>M(C3N+6 C3)2][>M(C3N6C3)2]− (I−)10

(LC20, Fig. 2(g)).67

To circumvent the shortcoming of weak absorption oflight at visible wavelengths, a variety of highly fluores-cent donor chromophore antennae have been covalentlyattached to the fullerene, such as porphyrins.72 Fullerene-porphyrin hybrids are more efficient in terms of singletoxygen generation and also have improved cell penetra-tion. Dialkyldiphenylaminofluorene (DPAF-C2M , shown in

Fig. 2(h)) is also a light-harvesting donor chromophoreantenna that can be attached to the C60 cage to facili-tate ultrafast intramolecular energy- and electron-transferfrom the donor antenna to C60 and can therefore be usedto enhance PDT efficacy.73 DPAF-C2M was constructed tohave increased optical absorption at 400 nm and also pos-sessed good two-photon absorption (2PA) cross-sectionsin the NIR wavelengths. Later another set derivativesC60(>CPAF-C2M� (Fig. 2(i)) was formed by structuralmodification via chemical conversion of the keto groupin C60(>DPAF-C2M) to a stronger electron-withdrawing1,1-dicyanoethylenyl (DCE) unit. This structural modifi-cation induced a large bathochromic shift of ground-stateabsorption of CPAF-C2M moieties beyond 450–550 nmand an increased electronic polarization of the molecule.The modification also led to a large bathochromic shiftof the major band in visible spectrum giving measureableabsorption up to 600 nm and extended the photoresponsivecapability of C60–DCE–DPAF nanostructures to longer redwavelengths than C60(>DPAF-C2M).

It was reported that the majority of the HOMO elec-tron density was delocalized over the dialkyldipheny-laminofluorene (DPAF-Cn) moiety, whereas the LUMOelectron density was located on the C60 spheroid.74

Therefore charge-separated states may be generated byintramolecular electron-transfer between the dipheny-laminofluorene donor and C60 > acceptor moieties duringthe photoexcitation process.Intramolecular formation of transient charge-separated

states is crucial for generation of radical ROS, initiallywith O−•

2 and subsequently HO•. C−•60 (>CPAF+•-Cn) is

the most stable charge-separated state in polar solvents,

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Functionalized Fullerenes in Photodynamic Therapy Huang et al.

including H2O.74 In nonpolar solvents, the intramolecular

energy-transfer event that produces 3C∗60(>CPAF-Cn) tran-

sient state dominates with nearly 6-fold higher generationof singlet O2 compared to 3C∗

60(>DPAF-Cn).

Examples of the Synthesis of Monocationic andPolycationic Fullerene DerivativeAs mentioned above cationic functional groups are gen-erally considered as the addend of choice for attachmenton the fullerene cage due to their potential surface bindingcontact with anionic residues of the bacteria cell wall viastatic charge interactions. A systematic trend to increasethe number of positive charges per fullerene cage wasdescribed in a recent report75 to maximize such interac-tions and use them as the approach for targeting bacte-ria having a significant density of anionic residues at thecell wall surface. A number of chemical functionalization

N N

NN

BF4 BF6

N

BF24

N N

N

N

N

N N

NN

orH2C=O

refluxtoluene

CH3I CH3I

+

OH

ON

H

(1.0 or 3.0 equiv.)

refluxtoluene

+

(1.0 or 2.0 equiv.)

N

N O

OH

H

H

N CH=O

N

BF22

N N

NN

H N

N

NH

N

NNH N

or

CH3I

CH3I

Figure 3. Synthesis of monocationic and tricationic dimethylpyrrolidinium [60]fullerenes BF4 and BF6, respec-tively, and mono- and bis(piperazinopyrrolidinium) [60]fullerenes BF22 and BF24, respectively.

methods of fullerenes have been reviewed.64�76–78 Amongthem, common convenient methods for the preparation ofcationic fullerene derivatives include cyclopropanation79

and pyrrolidination80 reactions due to their high con-sistency allowing product reproducibility. Examples ofthe latter were given in the preparation of quaternizeddimethylpyrrolidinium [60] fullerenyl monoadduct (BF4)and trisadduct (BF6).81�82 In a typical reaction condition,C60 was treated with 1.0 or 3.0 equivalent of N -methyl-glycine (sarcosine) and paraformalaldehyde in toluene atthe reflexing temperature to afford either mono-N -methyl-pyrrolidino[60] fullerene (BF4) or a large number of regioi-somers of tris(N -methylpyrrolidino)[60] fullerene (BF6)derivatives, as shown in Figure 3. Upon quaternization ofthese intermediates using methyl iodide as the methylationagent, corresponding monocationic and tricationic productsas BF4 and BF6 were obtained, respectively.

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HN

ON N N N N

HO

C3N6C3-OH

H2N N N N N N

N6C3

C70[>M(C3N6+C3)2]

I

I I

IHN

ON

N NN NOO

O O II

II

IHN

ONNNNN

I

C70[>M(C3N6+C3H)2]

HN

ON

N NN NOO

O O H

H

H

H

HHN

ONNN

NN

H

H

H

H

H

(CF3CO2 )5 (CF3CO2 )5

C70[>M(t-Bu)2]

OO

O O

C60[>M(C3N6+C3)2]

I

I I

IHN

ON N N

N NOO

O O II

II

IHN

ON

NNNN

I

Figure 4. Synthetic scheme for the preparation of C60[>M(C3N+6 C3)2] and C70[>M(C3N

+6 C3)2].

Similarly, the reaction of C60 in toluene witheither 1.0 or 2.0 equivalent of azomethine ylideproduced by piperazine-2-carboxylic acid dihydrochlo-ride dissolved in methanol and triethylamine in thepresence of 4-pyridinecarboxaldehyde at the refluxingtemperature gave the corresponding mono-piperazino-pyrrolidino[60] fullerene or a number of regioisomersof bis(piperazinopyrrolidino)[60] fullerene derivatives.Quaternization of both intermediates with methyl iodide ledto the corresponding monocationic and dicationic productsas BF22 and BF24 (Fig. 3), respectively.In the case of cyclopropanation reaction of C60 as

the functionalization method, it was applied recently forthe attachment of a highly complex decacationic moi-ety to the fullerene cage leading to the formation ofC60[>M(C3N

+6 C3)2] and C70[>M(C3N

+6 C3)2].

83 In thisreaction, a malonate precursor arm was applied to includetwo esters and two amide moieties, for a sufficientnumber of carbonyl and –NH groups in a short lengthof ∼ 20 Å, with a well-defined water-soluble pentaca-tionic moiety N+

6 C3 at each side of the arm for makingeffective multi-binding sites to the cell wall. The pre-cursor N+

6 C3 was reported to be a common synthon

for the structural modification of PDT nanomedicines.It was derived from the quaternization of N ,N ′,N ,N ,N ,N -hexapropyl-hexa(aminoethyl)amine precursor N6C3. Thebest method for the preparation of C60[>M(C3N

+6 C3)2]

and C70[>M(C3N+6 C3)2] was depicted in Figure 4

to begin with a well-defined fullerene monoadductderivatives, such as di(tert-butyl)fullerenyl malonatesC60[>M(t-Bu)2] and C70[>M(t-Bu)2], respectively, fol-lowed by facile transesterification reaction with thewell-characterized tertiary-amine precursor arm moiety,4-hydroxy-[N ,N ’,N ,N ,N ,N -hexapropyl-hexa(aminoethyl)butanamide (C3N6C3-OH) using trifluoroacetic acid asthe catalytic reagent to afford protonated quaternaryammonium trifluoroacetate salt C70[>M(C3N

+6 C3H)2].

Conversion of this salt to C70[>M(C3N+6 C3)2], was

accompanied by neutralization of trifluoroacetic acidby sodium carbonate and subsequent quaternization bymethyl iodide to give decacationic quaternary ammo-nium iodide salts. A similar conversion procedure wasapplied for the case of C60[>M(C3N

+6 C3)2]. This synthe-

sis represented the first examples of decacationic fullerenemonoadducts to incorporate a well-defined high number ofcation without the use of multiple addend attachments to

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Functionalized Fullerenes in Photodynamic Therapy Huang et al.

Figure 5. Synthesis of hexaanionic hexa(sulfo-n-butyl)-C60

(FC4S).

preserve the intrinsic photophysical properties of fullerenecages.

Synthesis of Hexa-Anionic Fullerene DerivativesFullerene molecules are highly hydrophobic. Pristine C60

can be dispersed into aqueous medium in a micelle formwith the application of surfactants. However, the micellestructure may not be stable enough in biological environ-ment. In a recent report, a strategy of creating a micelleformed from surfactants covalently bonded directly ontothe fullerene cage was illustrated by the synthesis ofhexa(sulfo-n-butyl)-C60 (FC4S) leading to structurally sta-ble molecular micelles in H2O.

84 The synthesis involvedthe use of hexaanionic C60 (C6−

60 ) chemistry85 for attach-ing six sulfo-n-butyl arms on C60 in one-pot reaction, asshown in Figure 5.It was found that molecular self-assemblies of FC4S

resulted in the formation of nearly monodispersespheroidal nanospheres with the sphere radius of gyrationRg ≈ 19 Å, where the major axe ≈ 29 Å and the minor axe≈21 Å for the ellipsoid-like aggregates, or an estimatedlong sphere diameter of 60 Å [the radius = (5/3)1/2Rg]for the aggregates, as determined by small angle neutronscattering (SANS) in D2O and small angle X-ray scatter-ing (SAXS) in H2O.

86 This radius of gyration was foundto remain relatively constant over a concentration rangefrom 0.35 to 26 mM in H2O, revealed strong hydrophobicinteraction between core fullerene cages overcoming loosecharge repulsion at the surface of the molecular micelle.It allowed the nanosphere formation at a low concen-tration despite of steric hindrance and high hydrophilic-ity arising from of 6 sulfo-n-butyl arms surrounding C60.Based on the SANS data, the mean number of FC4Smolecules for the nanosphere was determined to be 6�5±0�7 that led to the elucidation of its nanocluster structurewith each FC4S molecule located at the vertex of an octa-hedron shaped nanosphere shown in Figure 6.85

Synthesis of Chromophore-LinkedFullerene DerivativesOptical absorption of C60 is strong in UVA andweak in most of visible range. To circumvent thisshortcoming, a reported approach was described to use

Figure 6. (A) Structure of hexa(sulfo-n-butyl) [60]fullerene(FC4S) and (B) a characterized FC4S-derived nanosphereformed in H2O discussed by Yu.85

the light-harvesting donor chromophore antenna attach-ment at a very close vicinity of C60 cage, within a contactdistance of 2.6–3.5 Å, to facilitate ultrafast intramolecu-lar energy- and electron-transfer from the donor antennato C60 for enhancing PDT efficacy.73 A specific donorantenna, namely, dialkyldiphenylaminofluorene DPAF-Cn

was first introduced to give an increased optical absorp-tion at 400 nm and later being modified by replacingthe keto moiety of DPAF-Cn via a highly electron-withdrawing 1,1-dicyanoethylenyl (DCE) bridging groupthat resulted in dark burgundy-red C60(>CPAF-Cn) deriva-tives. This structural modification was found to inducea large bathochromic shift of ground-state absorption ofCPAF-Cn moieties beyond 450–550 nm.Preparation of C60(>CPAF-C2M�, as an example,

was made by Friedel-Craft acylation of 9,9-dimethoxy-ethyl-2-diphenylaminofluorene with bromoacetyl bromidein the presence of AlCl3 to yield 7-bromoactyl-9,9-dimethoxyethyl-2-diphenylaminofluorene, followed bycyclopropanation reaction with C60, as shown in Figure 7.The resulting product C60(>DPAF-C2M) was then furthertreated with malononitrile and pyridine in the presence oftitanium tetrachloride in dry toluene to yield C60(>CPAF-C2M) after chromatographic purification.

Photochemistry and Photophysics of FullerenylMolecular Micelles and Chromophore-FullereneConjugatesPhotoexcitation of C60 and fullerene derivatives induces asinglet fullerenyl excited state that is transformed to the

Figure 7. Synthetic method of C60(>CPAF-C2M) discussed byChiang.73

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Huang et al. Functionalized Fullerenes in Photodynamic Therapy

corresponding triplet excited state, via intersystem energycrossing, with nearly quantitative efficiency.41 Subsequentenergy transfer from the triplet fullerene derivatives tomolecular oxygen produces singlet molecular oxygen inaerobic media. This photocatalytic effect becomes oneof key mechanisms in photodynamic treatments usingfullerene derivatives as photosensitizers. However, a highdegree of functionalizaton on C60 for the enhancementof solubility and compatibility in biomedia resulted ina progressive decrease of the singlet oxygen produc-tion quantum yield [��1O2)]. Examples were given byBingel-type malonic acid, C60[C(COOH)2]n, and malonicester, C60[C(COOEt)2]n, [60] fullerene adducts,87 showinga decreasing trend of ��1O2) as the number of addends (n)increases. When the number n reached 6 for a hexaadduct,its � (1O2) value declined to only 13% or less of that forC60.

88 However, it was not the case for molecular micellarFC4S, a relatively high singlet oxygen production quan-tum yield for FC4S may indicate its unique electronic fea-tures in difference with Bingel-type malonic hexaadductsof C60.

85 The efficiency was substantiated by direct detec-tion of 1O2 emission at 1270 nm upon photoirradiation ofself-assembled FC4S nanospheres at 500–600 nm.

In the cases of light-harvesting electron-donor chro-mophore assisted fullerene conjugate systems, suchas C60(>CPAF-Cn) derivatives, their photophysicalproperties involve the primary photoexcitation eventsof either the fullerene moiety at UV wavelengths orthe DPAF-Cn moiety at both UV and visible wave-lengths up to 600 nm.73 Much higher optical absorptioncapability of DPAF-Cn than the C60> cage in visiblewavelengths enables the former moiety to serve as a light-harvesting antenna. Accordingly, formation of the pho-toexcited 1(DPAF)∗-Cn moiety should be considered as theearly event in the photophysical process. Alteration of theketo group of C60(>DPAF-Cn) to the 1,1-dicyanoethylenylgroup of C60(>CPAF-Cn) effectively extended its photore-sponsive region to longer red wavelengths. Photoexcitationprocesses of C60(>DPAF-Cn) and C60(>CPAF-Cn) pumpan electron from their highest occupied molecular orbital(HOMO) to the lowest unoccupied molecular orbital(LUMO). By the molecular orbital calculation and energyminimization, the majority of the HOMO electron densitywas reported to be delocalized over the dialkyldipheny-laminofluorene (DPAF-Cn) moiety, whereas the LUMOelectron density was located on the C60 spheroid, andtherefore C−•

60 (>CPAF+•-Cn) was suggested as the moststable charge-separated (CS) state in polar solvents, includ-ing H2O.

74 These charge-separated states may be generatedby photoinduced intramolecular electron-transfer betweenthe diphenylaminofluorene donor and C60> acceptor moi-eties. The process effectively quenches fluorenyl fluores-cence that can be observed in the most of C60(>DPAF-Cn)and C60(>CPAF-Cn) monoadducts. Even during energy-transfer events of C60(>CPAF-Cn), normally favorable in

non-polar solvents, observed short fluorescence lifetime ofthe model compound 1CPAF∗-C9 (241 ps) as comparedwith that of the keto analogous Br-1DPAF∗-C9 (2125 ps)may be indicative of a facile photoinduced intramolecu-lar charge polarization process forming the correspond-ing [C C(CN)2]

−•–DPAF+•-C9 charge-separated state thatwill facilitate the formation of C−•

60 (>CPAF+•-Cn) in thesubsequent electron-transfer event.

ANTICANCER EFFECT OF FULLERENE-PDTThere have been various studies demonstrating fullereneinduced in-vitro phototoxicity in cells. It is consideredthat one condition for any PS to produce cell killingafter illumination, is that the PS should really be takenup inside the cell, as the production of ROS outside thecell will not be enough to produce cell death unless it isproduced in extremely large amounts. One of the lim-itations for the study of the uptake of fullerenes intocells is their non-fluorescent nature that limits the useof fluorescence microscopy to study the localization incells. Some strategies though have been adopted to over-come this limitation, such as the use of radiolabeledfullerene that has been prepared to study the uptake.Indirect immunofluorescence staining with antibodies hasbeen used to show the localization of fullerene in mito-chondria and other intracellular membranes.89 Recentlyenergy-filtered transmission electron microscopy and elec-tron tomography was used to visualize the cellular uptakeof pristine C60 nanoparticulate clusters in the plasma mem-brane, lysosomes and in the nucleus of cells.90

In Vitro Anti-Cancer PDT with FullerenesThe first report of phototoxicity in cancer cells medi-ated by fullerenes was in the year 1993. In this studyTokuyama et al.91 used carboxylic acid functionalizedfullerenes at 6.0 �M and white light to produce growthinhibition in cancer cells. Burlaka et al.92 used pristineC60 at 10 �M with visible light from a mercury lampto produce some phototoxicity in Ehrlich carcinoma cellsor in rat thymocytes. The cytotoxic and photocytotoxiceffects of two water-soluble fullerene derivatives, a den-dritic C60 monoadduct and the malonic acid C60 trisadductwere tested on Jurkat cells when irradiated with UVA orUVB light.93 The cell death was mainly caused by mem-brane damage and it was UV dose-dependent.New approaches have been tested to overcome the

requirement to utilize UV or short-wavelength visiblelight to activate fullerenes. In one study where two newfullerene-bis-pyropheophorbide-a derivatives were pre-pared: a mono-(FP1) and a hexaadduct (FHP1). The C60

hexaadduct FHP1 had a significant phototoxic activity(58% cell death, after a dose of 400 mJ/cm2 of 688 nmlight) but the monoadduct FP1 had a very low photo-toxicity and only at higher light doses.94 Neverthelessthe activity of both adducts was less than that of pure

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Functionalized Fullerenes in Photodynamic Therapy Huang et al.

pyropheophorbide-a, possibly due to the lower cellularuptake of the adducts.95

The hypothesis that fullerenes have the potential todestroy cancer cells by PDT was tested in our group. Wehave shown that the C60 molecule mono-substituted with asingle pyrrolidinium group (BF4 shown in Fig. 2(c)) is anefficient PS and can mediate killing of a panel of mousecancer cells.20 The cells lines used were lung cancer (LLC)and colon cancer (CT26) adenocarcinoma and reticulumcell sarcoma (J774) and the latter showed much higher sus-ceptibility to fullerene mediated phototoxicity possibly dueto having an increased uptake because J774 cells behavelike macrophages. Besides the exceptionally active BF4,the next group of compounds has only moderate activity(BF2, BF5, and BF6 shown in Fig. 2(d)) against J774 cellsshowing only some killing even at high fluences, whilelast two compounds (BF1 and BF3) had no detectablePDT killing up to 80 J/cm2. The indirect measurementof fullerenes uptake was demonstrated by increase in flu-orescence of an intracellular probe (H2DCFDA) whichis specific for the formation of ROS. We also showedthe initiation of apoptosis by PDT mediated by BF4and BF6 in CT26 cells at 4−6 h after illumination.The induction of apoptosis was rapid after illuminationwhich may perhaps suggest that fullerenes are localizedin mitochondria, as it has been previously shown withbenzoporphyrin derivative.96–98 The explanation for themono-pyrrolidinium substituted fullerene as most effec-tive PS most likely linked to its relative hydrophobicity asestablished by its log P value of over 2. Besides this a sin-gle cationic charge on BF4 is in addition expected to playa significant role in determining its relative phototoxicity.Elisa Milanesio et al.97 used tetrapyrrole-fullerene con-

jugates and evaluated PDT effect with a porphyrin-C60

dyad (P-C60) and its metal complex with Zn(II) (ZnP-C60�and compared with 5-(4-acetamidophenyl)-10,15,20-tris(4-methoxyphenyl)porphyrin (P) on Hep-2 human larynx car-cinoma cell line. The phototoxicity was dependent on lightexposure level with visible light. 80% phototoxicity wasobserved for P-C60 after 15 min of light irradiation whichwas higher as compared to ZnP-C60. In case of argon atmo-sphere also a high photoactivity was observed with boththe dyads. In another paper,99 the cell death was confirmedto occur by apoptotic mode.As fullerenes show a relatively slower uptake we incu-

bated the cells for 24h with C60[>M(C3N+6 C3)2]-(I

−)10(LC14) and C70[>M(C3N

+6 C3)2]-(I

−)10 (LC17) (shownin Figs. 2(a) and (b)). C70[>M(C3N

+6 C3)2]-(I

−)10 killedcells more effectively than C60[>M(C3N

+6 C3)2]-(I

−)10, Onthe contrary, the fullerene drug LC14 killed less than1 log at all fluencies. LC17 that was short of the deca-tertiary amine chain was less phototoxic than LC18 whichpossessed an extra deca-tertiary ethyleneamine chain.This exciting result prompted us to carry out studieswith new PDT compounds C84O2[>M(C3N

+6 C3)2]-(I

−)10

(LC19) and C84O2[>M(C3N+6 C3)2][>M(C3N6C3)2]-(I

−)10(LC20)67 (shown in Figs. 2(f) and (g)). Different wave-lengths were used for irradiation. UVA and blue lightcaused more killing with LC20 than with LC19. This dif-ference can be attributed to better chance of electron trans-fer process occurring with shorter wavelengths and alsothe presence of the electron donating tertiary-polyethylene-amine chain. While when white light was employed thevariation between LC20 and LC19 was smaller but LC20still gave extra killing, while green light gave equivalentkilling for the two fullerenes. The situation was upturnedand LC19 gave considerably more killing than LC20 whenred light was used. It is important to state that the com-pounds used here induced a very low dark toxicity.

In Vivo Photodynamic Therapy of CancerThe three prerequisites for the fullerene PS to have pho-todynamic effect on tumors are first of all it should accu-mulate in the tumor tissue; secondly there should be apractically efficient way to administer the compound totumor bearing animals; and thirdly a practical way todeliver excitation light to the tumors.100 The first chal-lenge in this direction was taken up by Tabata56 in 1997.To make the water-insoluble pristine C60 water solubleand enlarge its molecular size they chemically modifiedit with polyethylene glycol. This conjugate was injectedintravenously into mice carrying a subcutaneous tumor onthe back. C60-PEG conjugate demonstrated higher accumu-lation and relatively more prolonged retention in the tumortissue than in normal tissue. On performing PDT afterintravenous injection of C60-PEG conjugate or Photofrin totumor-bearing mice, coupled with exposure of the tumorsite to visible light, the volume increase of the tumor masswas suppressed and the C60_PEG conjugate exhibited astronger suppressive effect than Photofrin. Tumor necrosiswas observed without any damage to the overlying nor-mal skin. The antitumor effect of the conjugate showed anincrease with increasing fluence delivered and C60 dose,and cures were achieved by treatment with a low dose of424 �g/kg at a fluence of 107 J/cm2. In another studyLiu and others55 conjugated polyethylene glycol (PEG)to C60 (C60-PEG), and diethylenetriaminepentaacetic acid(DTPA) was subsequently introduced to the terminal groupof PEG to prepare C60-PEG-DTPA that was mixed withgadolinium acetate solution to obtain Gd3+-chelated C60-PEG-DTPA-Gd. PDT induced anti-tumor effect and MRItumor imaging was evaluated on intravenous injection ofC60-PEG-DTPA-Gd into the tumor bearing mice. Equiv-alent generation of superoxide upon illumination wasobserved with or without Gd3+ chelation. Intravenousinjection of C60-PEG-DTPA-Gd into tumor bearing miceplus light (400∼500 nm, 53.5 J/cm2) demonstrated sig-nificant anti-tumor PDT depending on the timing of lightirradiation that also correlated with tumor accumulation asdetected by the enhanced MRI signal.

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Figure 8. (A) Bioluminescence imaging of CT26-Luc tumors growing in a representative control mouse (upper panel) and arepresentative IPPDT treated mouse (lower panel). (B) Quantitative analysis of bioluminescence dynamics in control and whitelight treated mice (n = 10 per group). Reprinted with permission from [102], P. Mroz, et al., Intraperitoneal photodynamic therapymediated by a fullerene in a mouse model of abdominal dissemination of colon adenocarcinoma. Nanomedicine 7, 965 (2011).© 2011, Future Science.

Yu and her coworkers101 performed a preliminary in vivostudy of PDT using hydrophilic nanospheres formed fromhexa(sulfo-n-butyl)-C60(FC4S, shown in Fig. 3(A)). Thisstudy was performed on ICR mice bearing sarcoma180 subcutaneous tumors. No adverse effects were notedin the animals when the FC4S was administered orally.Water-soluble FC4S in PBS (5 mg/kg body weight) wasgiven either intraperitoneally or intravenously with subse-quent irradiation with an argon ion laser beam at a wave-length of 515 nm or an argon-pumped dye-laser at 633 nm.The beam was focused to a diameter of 7–8 mm with thetotal light dose of 100 J/cm2. Inhibition of tumor growthwas found more effective using the low wavelength i.e., incase of better-absorbed 515nm laser than the 633 nm laser.I ·p. administration method proved to be slightly better ininhibition effectiveness than the i.v. method. Conclusivelydata suggest that PDT with fullerenes is not only possiblein animal tumor models, but can demonstrate the potentialuse of these compounds as PS for PDT of cancer.We have also recently shown102 the therapeutic effects

of intraperitoneal PDT with fullerene and white light ina very challenging mouse model of disseminated abdom-inal cancer. In this study we prepared the monocationicBF4 (Fig. 2(c)) in micelles composed of Cremophor EL.Colon adenocarcinoma cell line (CT26) expressing fire-fly luciferase was used to allow monitoring of IP tumorburden by non-invasive biolumi8nescence imaging. BF4 inmicelles was injected intraperitonally (5 mg/kg) followedby white-light illumination (100 J/cm2) delivered throughthe peritoneal wall. This produced a statistically significantreduction in bioluminescence and besides this produced asurvival advantage in mice, shown in Figure 8. A drug-light interval of 24 h was more effective than a 3 h drug-light interval showing the significance of allowing enoughtime for the fullerene to be taken up into the cancer cells.As the cancer cells are known to express more glucose

receptors, Otake et al. exploited this fact and synthesizedgroup of C60-glucose conjugates which also proved to

be more soluble. These conjugates demonstrated selectivephototoxicity compared to fibroblast cells thus suggest-ing the significance of targeting glucose receptors.103 ThePDT effect in vivo was investigated in human-melanoma(COLO679)-xenograft bearing mice by injecting C60-(Glc)1 (0.1 or 0.2 mg/tumor) intratumorally followed byirradiated with 10 J/cm2 UVA 1ight. The drug-light inter-val was 4 h. Tumor growth was suppressed better with thehigher dose than the lower dose.

ANTIMICROBIAL EFFECT OF FULLERENESAntibiotic resistance is a worldwide problem that isspreading with remarkable speed. The injudicious andover-use of antibiotics is the most important reason leadingto antibiotic resistance around the world.The “golden age” of antimicrobial therapy began with

the discovery of antibiotics around the middle of thelast century.104 Meanwhile many other ancient effectiveantibacterial treatments including photosensitizing reac-tions were forgotten. In the last few decades, however,the widespread use of antimicrobial agents emerges theincrease of antibiotic-resistant bacteria and other infectiousmicroorganisms and led to predictions of untreatable infec-tions caused by “superbugs,”105 which in turn has createdan ever-increasing need for new drugs.Therefore antimicrobial PDT has become an emerging

alternative strategies for destroying microorganisms espe-cially for multi-drug resistant pathogens.106 PDT producesROS that are toxic to the target microorganisms. PDT hasa broad spectrum of action, and compared to antibiotictreatment PDT does not lead to the selection of mutantresistant strains.Currently, topical application of a PS on infected tis-

sues and subsequent illumination seems to be the mostprominent feature of antimicrobial PDT, without damagingthe surrounding tissue or disturbing the residual bacterial-flora. It was well accepted that Gram-positive bacteria are

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Functionalized Fullerenes in Photodynamic Therapy Huang et al.

more susceptible to PDT as compared to Gram-negativebacteria. This can be explained by the different structuresof their cell walls.107

There are several possible mechanisms to explain theantimicrobial activity of illuminated fullerene PS: by inter-fering with cell wall synthesis; plasma membrane integrity;nucleic acid synthesis; ribosomal function and folate syn-thesis. All of these would result in disruption of the bac-terial cell function and inhibit their growth.Martin and Logsdon hypothesized that it was pos-

sible that microorganisms were susceptible to damageby to Type I ROS when compared with Type II sin-glet oxygen.108 As mentioned before fullerenes can gainsolubility63 and produce more hydroxyl radicals andsuperoxide anion, as well as singlet oxygen throughfunctionalization109 by attaching some hydrophilic oramphiphilic functional groups.22

An ideal PS proposed for antimicrobial PDT can bejudged on several criteria. These PS should have no tox-icity in the dark and should selectively kill bacteria overmammalian cells. PS should be able to kill multiple classesof microorganisms at relatively low concentrations withlow fluences of light. PS should ideally have high absorp-tion around 600 nm to 800 nm and generate high tripletand singlet oxygen quantum yields.

Antimicrobial Effects In VitroThe structures, especially the charges of attached groupson fullerene influence the efficacy of PS on killingmicroorganisms. Our lab has shown, in a series of reportedexperiments that cationic fullerenes fulfill many, but notall of the aforementioned criteria. At the first time wedemonstrated that the soluble functionalized fullereneswere efficient antimicrobial PS and could mediate selec-tive photodynamic inactivation (PDI) for various classesof microbial cells over mammalian cells.75 We comparedthe antimicrobial activity of broad-spectrum antimicrobialphotodynamic activities of two series of functionalizedC60; a first series with one, two, or three polar diseri-nol groups, and a second series with one, two, or threequarternary pyrrolidinium groups. Gram-positive bacteria(S. aureus) Gram-negative bacteria (E. coli and P. aerug-inosa), and fungal yeast (C. albicans) were tested in thisstudy. The neutral, alcohol-functionalized fullerenes hadonly modest activity against S. aureus, while the cationicpyrrolidinium-functionalized fullerene (BF6, Fig. 2(d))was surprisingly effective in causing light-mediated killingof S. aureus at 1 �M, and 10 �M for E. coli, C. albicansand P. aeruginosa. However, the pyrrolidinium- functional-ized fullerenes compound BF6 demonstrated high levels ofdark toxicity against S. aureus, Mashino et al. showed thatcationic fullerenes could inhibit the growth of E. coli andS. aureus by interfering with the respiratory chain.110�111

This data suggests that photoactivated fullerenes may havesomewhat different sites of action in bacteria compared to

more traditional PS such as tetrapyrroles that generate sin-glet oxygen. These compounds all performed significantlybetter than a widely used antimicrobial photosensitizer,toluidine blue O.In agreement with previous discussion, results from

Spesia et al.112 indicated that a dicationic fullerene deriva-tive was an interesting PS with potential applicationsin PDI of bacteria. They compared the PDI efficacy offullerene derivatices with different numbers of cationiccharges. The spectroscopic and photodynamic propertiesof a dicationic N�N -dimethyl-2-(40-N ,N ,N -trimethyl-aminophenyl) fulleropyrrolidinium iodide) (DTC2+

60 ) werecompared with a non-charged N -methyl-2-(4′-acetamido-phenyl)fulleropyrrolidine (MAC60) and a monocationicN ,N -dimethyl-2-(4′-acetamidophenyl)fulleropyrrolidiniumiodide (DAC+

60) in different media and in a typicalGram-negative bacterium, E. coli. PDI of E. coli cellularsuspensions by dicationic fullerene exhibits a ∼ 3.5 logdecrease of cell survival after 30 min of irradiation, whichrepresents about 99.97% of cellular inactivation.To determine the optimal chemical structures produced

by fullerene derivatization, a QSAR relationship studyemployed fullerene PS with a wider range of differenthydrophobicities, as well as with an increased number ofcationic charges.113 The results indicated that increasing thenumber of cationic charges and lowering the hydrophobic-ity tended to increases the antimicrobial PDI efficacy offullerene PS against both Gram-positive and Gram-negativebacteria. The charge increases the association of the PSwith negatively charged pathogen membranes, whereas thehydrophobic character increases association with or pen-etration into the lipid components of the membrane, orboth. Recently, Mizuno et al.82 from our laboratory empha-sized the importance of the number of cationic charges ininfluencing the efficiency of the fullerenes in antimicrobialPDI when they looked at a further series of functionalizedcationic fullerenes PS. They compared PDI efficacy of anew group of synthetic fullerene derivatives that possessedeither basic or quaternary amino groups as antimicrobial PSagainst S. aureus (Gram-positive), E. coli (Gram-negative)bacteria and C. albicans (fungi). QSAR derived with Log Pand hydrophilic lipophilic balance parameters showed thatmuch better correlations were obtained when 3× the num-ber of cationic charges were subtracted from the Log P val-ues. The most effective ones to perform antimicrobial PDTwere tetracationic compound BF21 that had more cationiccharges and a lower log P. S. aureus was most susceptible;E. coli was intermediate, while C. albicans was the mostresistant species tested.Antimicrobial effect of two highly water-soluble deca-

cationic fullerenes LC14 (C60[>M(C3N+6 C3)2]) was and

LC17 (C70[>M(C3N+6 C3)2]) were applied for compari-

son in the PDT-killing of the Gram-positive S. aureus.66

The decacationic arms attached to these fullerenes affili-ated the rapid binding to the anionic residues of bacterial

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Huang et al. Functionalized Fullerenes in Photodynamic Therapy

cell walls. The large number of ionic groups dramaticallyenhanced water solubility of these compounds. The datashowed interesting differences between the photoactivityof decacationic fullerene compounds that differ only inthe number of carbon atoms in the fullerene cage. ForGram-positive bacteria C60[>M(C3N

+6 C3)2] was better at

photokilling than C70[>M(C3N+6 C3)2], while for Gram-

negative bacteria and for cancer cells the opposite was thecase, in that C70[>M(C3N

+6 C3)2] was better at photokilling

than C60[>M(C3N+6 C3)2]. The results of ROS (HO• or

1O2) generation demonstrated that C60[>M(C3N+6 C3)2]

produced more 1O2 while C70[>M(C3N+6 C3)2] produced

more HO•. This finding offers an explanation of the pref-erential killing of Gram-positive bacteria by LC14 and thepreferential killing of Gram-negative bacteria by LC17.This finding is in agreement with our previous report thatType II ROS, i.e., singlet oxygen, 1O2, are better at killingGram-positive bacteria than Type I ROS, i.e., hydroxylradicals, HO•, while the reverse is true for Gram-negativebacteria (HO• is better at killing than 1O2). The hypothesisis that 1O2 can diffuse more easily into porous cell wallsof Gram-positive bacteria to reach sensitive sites, while the

Figure 9. BF6-PDT and tobramycin treatment of Pseudomona aeruginosa wound-infected mice. (A) Representative biolumines-cence images of P. aeruginosa-infected mice (captured immediately postinfection, immediately post-treatment and 24 h post-treatment), receiving: no treatment (top row); treated with BF6-PDT alone (180 J/cm2; second row); treated with Tobr alone(6 mg/kg for 1 day; third row, diagonal panel 24 h post-treatment shows two possible outcomes); and treated with a combinationof BF6-PDT and 1 day Tobr (bottom row). (B) Quantification of luminescence values from bioluminescence images (not shown)obtained during the PDT process, or at equivalent times for non-PDT mice. ∗p < 0�05; ∗∗p < 0�01; ∗∗∗p < 0�001; BF6 plus light(with and without Tobr) versus BF6 in dark and versus Tobr alone. (C) Kaplan–Meier survival curves for the groups of mice inFigure 4(A); no treatment control (n = 10); PDT alone (n = 12); Tobr alone (n = 2); PDT plus Tobr (n = 10). PDT: Photodynamictherapy; Tobr: Tobramycin. Reprinted with permission from [81], Z. Lu, et al., Photodynamic therapy with a cationic functionalizedfullerene rescues mice from fatal wound infections. Nanomedicine (Lond.), 5, 1525 (2010). © 2010, Future Science.

less permeable Gram-negative bacterial cell wall needs themore reactive HO• to cause real damage.114�115

Antimicrobial Effect In VivoThe absorption spectrum of fullerenes is, in additionto substantial UV absorption, mainly in the blue andgreen visible wavelengths. This property actually limitsthe application of fullerene in clinical disorders, once thepenetration of short wavelength light into tissue is rel-atively poor; however, fullerenes may still be useful asantimicrobial PS for the treatment of relatively superfi-cial infections, where the light does not need to pene-trate deeper than 1 mm. A fullerene-based PS (BF6) withtricationic charges provided by quaternized dimethylpyrro-lidinium groups was found to be an effective againstGram-positive bacteria, Gram-negative bacteria and fun-gal yeast in vitro.75 To investigate if the high degree ofin vitro activity could translate into an in vivo antibacterialPDT effect, our lab81 continued to test BF6 in two poten-tially lethal mouse models of wounds infected with twoGram-negative bacteria (P. aeruginosa and P. mirabilis),respectively. Compared to Gram-positive bacteria, many

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Functionalized Fullerenes in Photodynamic Therapy Huang et al.

Figure 10. Representative bioluminescence images frommice with Escherichia coli burn infections (day 0) and treatedwith successive fluences of photodynamic therapy or UVAlight alone. (A) UVA control; (B) LC17 + UVA light; and(C) LC18+UVA light. There was no significant reduction inbioluminescence after application of either LC17 or LC18without light exposure as a dark control. Reprinted withpermission from [119], L. Huang, et al., Antimicrobial photody-namic therapy with decacationic monoadducts and bisadductsof [70]fullerene: In vitro and in vivo studies. Nanomedicine(Lond.) (2013). © 2013, Future Science.

Gram-negative bacteria are much more difficult to bephoto-inactivated, and tend to produce systemic sepsisafter developing infections in wounds. Higher concentra-tions of PS and higher fluences of light (180 J/cm2) were

Figure 11. Representative bioluminescence images from mice with Acinetobacter baumannii burn infections and treated withphotodynamic therapy, UVA light alone or absolute control, captured day 0 (before photodynamic therapy) and then daily for 6days. (A) Absolute control; (B) UVA control+15% DMA; (C) LC17+15% DMA; (D) LC18+15% DMA; (E) LC17+15% DMA+UVAlight; and (F) LC18+15% DMA+UVA light. Reprinted with permission from [119], L. Huang, et al., Antimicrobial photodynamictherapy with decacationic monoadducts and bisadducts of [70]fullerene: In vitro and in vivo studies. Nanomedicine (Lond.) (2013).© 2013, Future Science.

needed in vivo than in vitro to achieve a certain loss ofbioluminescence. The fullerene-mediated PDT succeededin saving the life of mice whose wounds were infectedwith P. mirabilis and could be combined with a sub-optimal dose of antibiotics to save mice with P. aeruginosawound infections. These exciting results shown in Figure 9indicated that fullerene-mediated PDT could either treatwounds infected with virulent species of Gram-negativebacteria or be able to synergize with a suboptimal antibi-otic regimen to prevent regrowth and produce significantlyhigher survival.81

In the case of the 3rd-degree burns, they are particu-larly susceptible to bacterial infection as the barrier func-tion of the skin is destroyed, the dead tissue is devoid ofhost-defense elements, and a systemic immune suppres-sion is a worrying consequence of serious burns. Further-more, the lack of perfusion of the burned tissue means thatsystemic antibiotics are generally ineffective.116 Althoughexcision and skin grafting is now standard treatment forthe 3rd-degree burns,117 superimposed infection is still amajor problem. Patients with Gram-negative burn infec-tions have a higher likelihood of developing sepsis thanGram-positive infections. Topical antimicrobials are themainstay of therapy for burn infections and PDT may havea major role to play in the management of this disease.118

We found in previous study a decacationic fullereneLC17 (C70[>M(C3N

+6 C3)2]) was effective at mediating

the photokilling of Gram-negative bacteria in vitro.66

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Huang et al. Functionalized Fullerenes in Photodynamic Therapy

We synthesized a new compound C70[>M(C3N6+C3)2]

[>M(C3N6C3)2] (LC18) with the same decacationic sidechain plus an additional deca-tertiary amine groups againstGram-negative bacteria. A mouse model of the third-degree burn infection with bioluminescent Gram-negativebacteria was used to test the in vivo effectiveness of thetherapeutic approach using the UVA excitation.119 The datashown in Figures 10 and 11 suggested that the attach-ment of an additional deca(tertiary-ethylenylamino) mal-onate arm to C70, producing LC18, allowed the moietyto act as a potent electron donor and increased the gen-eration yield of hydroxyl radicals under UVA illumina-tion. This is consistent with the reported phenomena ofphotoinduced intramolecular electron transfer from the ter-tiary amine moiety to the fullerene cage in polar solvents,including water, at the short excitation wavelength. Withthe availability of ten tertiary amine moieties, each capa-ble of donating one electron to the C70 cage, LC18 mayfunction as an electron-rich precursor for hydroxyl radicalproduction that demonstrated a new approach in enhanc-ing HO• radical killing of pathogenic bacteria in contrastto the more common 1O2-killing mechanism.

CONCLUSIONFullerenes have been widely studied as potential PS thatcould mediate PDT of diverse diseases. As discussed previ-ously fullerene-derivatives have uniquely important favor-able properties and an unusual photochemical mechanism,which could make them candidates for ideal PS. As shownby us and by others, fullerene-derivatives produce a sub-stantial amount of superoxide anion in a Type I photochem-ical process involving electron transfer from the excitedtriplet state to molecular oxygen in aqueous biomedicalsolutions. It is assumed that hydroxyl radicals are formedfrom hydrogen peroxide, and hydroxyl radicals are the mostreactive and potentially the most cytotoxic of all ROS.The chief disadvantage of fullerenes is likely to be that

their absorption spectrum of fullerenes is highest in theUVA and blue regions of the spectrum, which limit thetissue penetration depth of illumination. With the correctfunctionalities present on the fullerene cage, these diffi-culties may be overcome. Since in vivo PDT usually usesred light for its improved tissue-penetrating properties it isunclear whether fullerenes would mediate effective PDTin vivo. Therefore synthesis of new fullerene derivativeswill be a trend for future study, particularly those withlight-harvesting antennae to broaden the absorption light,hence increasing light penetration depth into tissue. Fur-thermore 2-photon excitation is another promising avenueto increase penetration depth of PDT. The mechanisticstudy of Type I and Type II photochemistry, and the corre-lations between fullerene structure, photochemical mech-anism and PDT efficacy will establish whether fullerenescan compete with more traditional PS in clinical applica-tions of PDT.

Acknowledgments: Long Y. Chiang was supported byUS NIH grant R01CA137108. Michael R. Hamblin wassupported by US NIH grant R01AI050875.

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