text:
Review Journal of Biomedical Nanotechnology
Copyright © 2014 American Scientific Publishers All rights reserved Printed in the United States of America
Vol. 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 ∗ 1
Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA 02114, USA Department of Dermatology, Harvard Medical School, Boston, MA 02115, USA 3 Raja Ramanna Centre for Advanced Technology, Indore, MP 452013, India 4 Department of Dermatology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China 5 Department of Chemistry, Institute of Nanoscience and Engineering Technology, University of Massachusetts, Lowell, MA 01854, USA 6 Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139, USA 2
Since the discovery of C60 fullerene in 1985, scientists have been searching for biomedical applications of this most fascinating of molecules. The unique photophysical and photochemical properties of C60 suggested that the molecule would function well as a photosensitizer in photodynamic therapy (PDT). PDT uses the combination of non-toxic dyes and harmless visible light to produce reactive oxygen species that kill unwanted cells. However the extreme insolubility and hydrophobicity of pristine C60 , mandated that the cage be functionalized with chemical groups that provided water solubility and biological targeting ability. It has been found that cationic quaternary ammonium groups provide both these features, and this review covers work onDelivered the use of to mediate destruction bycationic Ingentafullerenes to: University of South Carolinaof cancer cells and pathogenic microorganisms in vitro and describes the treatment of tumors and microbial infections IP: 46.161.56.113 On: Mon, 23 May 2016 08:28:44 in mouse models. The design, synthesis, and use of simple pyrrolidinium salts, more complex decacationic chains, and light-harvesting antennae that Copyright: American Scientific Publishers can be attached to C60 , C70 and C84 cages are covered. In the case of bacterial wound infections mice can be saved from certain death by fullerene-mediated PDT.
KEYWORDS: Fullerene, Photodynamic Therapy, Reactive Oxygen Species, Cancer, Infections, Electron Transfer, Singlet Oxygen, Cationic Charge.
CONTENTS Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Traditional Photosensitizers . . . . . . . . . . . . . . . . . . . . . . Photophysics and Photochemistry of PDT . . . . . . . . . . . Fullerenes as Photosensitizers . . . . . . . . . . . . . . . . . . . . Photophysics and Photochemistry of Fullerenes and Derivatives . . . . . . . . . . . . . . . . . . . . . Design of Fullerene Derivatives . . . . . . . . . . . . . . . . . . . Examples of the Synthesis of Monocationic and Polycationic Fullerene Derivative . . . . . . . . . . . . . . . . Synthesis of Hexa-Anionic Fullerene Derivatives . . . . . . Synthesis of Chromophore-Linked Fullerene Derivatives . . Photochemistry and Photophysics of Fullerenyl Molecular Micelles and Chromophore-Fullerene Conjugates . . . . . . Anticancer Effect of Fullerene-PDT . . . . . . . . . . . . . . . . . In Vitro Anti-Cancer PDT with Fullerenes . . . . . . . . . . . In Vivo Photodynamic Therapy of Cancer . . . . . . . . . . . Antimicrobial Effect of Fullerenes . . . . . . . . . . . . . . . . . . ∗
Author to whom correspondence should be addressed. Email: [email protected] Received: 6 February 2014 Accepted: 5 March 2014
J. Biomed. Nanotechnol. 2014, Vol. 10, No. 9
. . . 1921 . . . 1922 . . . 1924 . . . 1926 . . . 1926 . . . . .
Antimicrobial Effects In Vitro Antimicrobial Effect In Vivo . Conclusion . . . . . . . . . . . . . . Acknowledgments . . . . . . . . References . . . . . . . . . . . . .
INTRODUCTION Photodynamic therapy (PDT) is based on the administration of nontoxic light absorbing dyes called photosensitizers (PS), either systemically, locally or topically, followed by irradiation of harmless visible to near infrared (NIR) light, in the presents of oxygen, leading to the generation of cytotoxic reactive oxygen species (ROS).1 Dual selectivity of PDT can be obtained by the therapeutic gradient of photosensitizer concentration between tumor and normal tissues and precise delivery of light exposure within the tumor.2 However, the major limitations of PDT are non-specific cellular uptake of PS by normal cells and short light penetation depth. PDT is a widely recognized valuable treatment option for neoplastic and non-malignant 1550-7033/2014/10/1918/019
doi:10.1166/jbn.2014.1963
Huang et al.
Functionalized Fullerenes in Photodynamic Therapy
diseases. Recently with the development of fiber-optic systems, light can be delivered accurately into many parts of the body for the treatment of tumors. Therefore, PDT applications have been expended to many endoscopically accessible tumors, including lung cancer, superficial gastric cancer, head and neck cancer, cervical cancer and bladder cancer. In general, there are a number of advantages for PDT over chemotherapy and radiotherapy: PDT shows no
long-term side effects when an effective PS is employed; it is a minimally-invasive procedure with little or no scarring after the site heals as compared to surgery; it can deliver highly targeted precision at the disease site; there can be repeatable treatments at the same site if needed; PDT can be less costly than other cancer treatments; it may take only a short period of time for each session allowing treatment as an outpatient. The main limitation of PDT
Ying-Ying Huang, M.D., is a researcher in Dr. Michael Hamblin's lab in Wellman Center for Photomedicine at Massachusetts General Hospital, an Instructor of Dermatology at Harvard Medical School. She received her M.D. from China in 2004. She earned 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 the areas of photodynamic therapy (PDT) for infections, cancer and mechanism of low level light therapy (LLLT) for traumatic brain injury. She has published 48 peer review articles and 15 conference proceedings and book chapters. She is the co-editor of newly released "Handbook of Photomedicine."
Sulbha K. Sharma, Ph.D. is a visiting fellow at Raja Ramanna centre for advanced technology, Indore, India. Earlier she was a postdoctoral fellow at Dr. Hamblin's lab at The Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA. She completed her Ph.D. from Laser Biomedical section and instrumentation division at Raja Ramanna centre for advanced technology, Indore, India. She has published 25 peer-reviewed articlesto:7 University conferenceofproceedings and 4 book chapters. Her research Delivered by Ingenta South Carolina IP: 46.161.56.113 Mon, 23 May 2016and 08:28:44 interests are anticancerOn: photodynamic therapy low level light therapy. Copyright: American Scientific Publishers
Rui Yin, M.D., Ph.D. is an Associate Professor of Dermatology, Southwest Hospital, Third Military Medical University, and a visiting associate professor of Wellman Center for Photomedicine at Massachusetts General Hospital. Her research interests lie in the areas of photodynamic therapy for infections and cancer, the electron transfer mechanisms of photodynamic reaction. She has published 17 peer-reviewed articles in 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 Science Foundation of China as a grant reviewer. She is also a committee member of China Dermatologist Association and China Medical Association. In 2011, Dr. Yin was honored as one of Top 10 National Outstanding Young Dermatologist by China Dermatologist Association. Tanupriya Agrawal, M.D., Ph.D. is a postdoctoral fellow at Dr. Hamblin's lab at The Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA. Prior to this, she finished medical school at N.S.C.B Medical College, Jabalpur, India followed by Ph.D. in Biomedical Sciences at Creighton University, Omaha, NE. She has completed United States Medical Licensing Examination (USMLE) and certified by Educational Commission for Foreign Medical Graduates (ECFMG). She has published 6 peer-reviewed articles and 12 conference proceedings. She is investigating the role and underlying molecular mechanisms of low level laser therapy in neurogenesis and synaptogenesis in mouse model of traumatic brain injury.
J. Biomed. Nanotechnol. 10, 1918–1936, 2014
Long Y. Chiang, Ph.D. is a Professor at the Department of Chemistry, University of Massachusetts Lowell. His research interests lie in the areas of design and synthesis of ultrafast broadband photoresponsive linear and nonlinear multiphoton energy absorptive fullerenyl nanostructures and polycationic, nano-PDT drugs in combination of photoenergy, 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 269 peer-reviewed articles, book chapters, review articles, and awarded 39 patents. He was a chairman of several MRS symposia and a member of international advisory committee of ICSM. He was a member of editorial board and regional editor of two journals and is for one journal. Michael R. Hamblin, Ph.D. is a Principal Investigator at the Wellman Center for Photomedicine at Massachusetts General Hospital, an Associate Professor of Dermatology at Harvard Medical School and is a member of the affiliated faculty of the Harvard-MIT Division of Health Science and Technology. His research interests lie in the areas of photodynamic therapy (PDT) for infections and cancer, and in low-level light therapy (LLLT) for wound healing, arthritis, traumatic brain injury and hair regrowth. He has published 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 journals and serves on NIH Study Sections. In 2011 Dr Hamblin was honored by election as a Fellow of SPIE.
is its action occurs only at areas where light can reach. (verteporfin, benzoporphyrin derivative monoacid ring A, BPD-MA; Novartis Pharmaceuticals), NPe6 (mono-LThis implies that the major sites of tumors treated by PDT by Ingenta South Carolina aspartylofchlorin e6, taporfin sodium, talaporfin, LS11; are the lining of organs or just Delivered under the skin that canto: be University IP: 46.161.56.113 On: Mon, 23 May 2016 08:28:44 Light Science Corporation), Levulan® (5-aminolevulinic reached by the light source. Copyright: American Scientific Publishers acid, a precursor of protoporphyrin IX),9 and phthalocyanines (Pc4).10 The term "third generation PS" refers to TRADITIONAL PHOTOSENSITIZERS the 2nd generation PS bound to carriers such as antibody Photosensitizers can be categorized by their chemiconjugates11 liposomes and other targeted structures for cal structures as porphyrins (tertrapyrroles) and nonincreasing the selectively for tumor tissues. Although the porphyrins.3 First, second or third generation PS are terms majority of PS are porphyrin derivatives, non-porphyrin further used to label tetrapyrrole-derived PS. Porphyrins PS are being employed to improve PDT efficacy and are derived from the tetrapyrrole aromatic macrocycle minimal side effects. Synthetic, non-naturally-ocurring, which is a major component of many naturally occuring conjugated or expanded pyrrolic ring systems are another pigments such as heme, chlorophyll and bacteriochloroclass of non-porphyrin PS, including texaphyrins, porphyll. Porphyrins contain a structure with a 22 -electron phycenes, phthalocyanines and naphthalocyanines. Other system which gives rise to their long wavelenth absorption compounds that have been studied as PS are not derived of light.4 Hematoporphyrin (Hp), hematoporphyrin devivafrom the tetrapyrrole backbone, but can be classed tive (HpD) and Photofrin are referred to as first generation as miscellaneous dyes including chalcogenopyrylium PS. HpD and Photofrin have been widely used in clinical dyes,12 phenothiazinium dyes including methylene blue for cancer.5 However, the side effects associated with 1st and toluidine blue,13 Nile blue derivatives,14 hydroxygeneration PS, such as prolonged skin photosensitiztion lated perylenequinones such as hypericin,15 BODIPY and suboptimal tissue penetation of the 630 nm light derivatives,16 squaraines17 etc. These compounds have have stimulated interst in development of new PS. Second provided a new focus to the field of PDT. generation of PS are chemically purified or synthetic It is generally accepted that the characteristics that the tetrapyrrole derivatives, which absorb longer wavelength ideal PS used against cancer should possess are: light and cause less skin photosensitization. Some promis(1) single compound with known composition and good ing second generation photosensitizers have been approved stability, or tested in clinical trials.6 These include, but are not (2) preferential uptake and retain in the target tumour limited to, palladium-bacteriopheophorbide (TOOKAD),7 tissue, meso-tetra-hydroxyphenylchlorin (Foscan® , Temoporfin),8 (3) minimal toxicity in the absence of light to prevent harmful side-effect to the surrounding normal tissue, tin-ethyletiopurpurin (SnET2, Purlytin), Visudyne® 1920
(4) high quantum yield of triplet state and ROS generation and (5) high molar extinction coefficient to minimize the dose of PS needed to achieve the desired PDT effect, (6) intrinsic fluorescence to permit their detection by optical imaging (microscopy) techniques, and (7) high absorbance, particularly in the red part of the spectrum, which leads to a deeper light penetration into the tissues; for instance, the light depth penetration at 500 nm is about 4 mm, whereas at the 600–800 nm range it is about 8 mm2 .
the option of attaching the light-harvesting antennae to the fullerenes to increase the quantum yield of production of ROS. Fullerenes can self-assemble into vesicles called fullerosomes that can act as multivalent drug delivery vehicles with the possibility of different targeting properties.21 Fullerenes have been said to offer "a wide open playing field to chemists"22 by providing synthetic opportunities to attach a wide variety of hydrophilic or amphiphilic side chains or fused-ring structures to the spherical C60 core.22 Furthermore, fullerenes have hollow interiors, where other atoms, ions or small clusters can be entrapped and form endohedral fullerenes. Those fullerenes that encapsulate Photophysics and Photochemistry of PDT metal atoms are called endohedral metallofullerenes.23 Most PS have 2 electrons with opposite spins located in Hydroxylation (attaching OH-groups) is the most coman energetically lower energy orbital, the so-called highmon functionalization, which renders the molecule more est occupied molecular orbital (HOMO), in their ground hydrophilic,24 but other polar adducts will also have the (usually single) state. Absorption of light (photons) leads same effect.25 to an excited singlet state by the elevation of one elecThere are also several unfavorable characteristics of tron with unchanged spin to a higher energy obital, fullerenes for PDT, but by applying different strategies called the lowest unoccupied molecular obital (LUMO).18 they can be overcome. The main absorption of fullerenes This excited singlet PS is short-lived (nanoseconds) and occurs in the UV, blue and green regions of the spectrum emits excess energy as fluorescence and/or heat. Alternawhile the absorption of tetrapyrrole PS (such as chlorins, tively, an excited PS may undergo an intersystem crossing bacteriochlorins, and phthalocyanines) shows substantial (ISC) by inverting spin of one electron to form a relpeaks in the red or far-red regions where the penetraatively long-lived (microseconds to milliseconds) triplet tion of light into tissue is much deeper. This major state. disadvantage of fullerenes however can be overcome in The excited triplet PS can either decay radiationlessly to University of South Carolina Delivered by Ingenta to: several ways, for example by chemical attachment of one the ground state or can survive long enough to transfer itsMon, 23 May 2016 08:28:44 IP: 46.161.56.113 On: or more red-wavelength absorbing antennae onto a C60 energy to molecular oxygen O2 (ground triplet state) and Scientific Copyright: American Publishers cage.26 The absorption of many light photons simulta1 produce excited state singlet oxygen ( O2 ). This reaction neously can be achieved by the attachment of multiple is referred to as a Type II process. A Type I process can light-harvesting antennae on one C60 cage. The unfavoralso occur whereby the PS reacts directly with neighborable absorption spectrum of fullerenes can also be overing molecules and gains or donates an electron to form come by using optical clearing agents to the tissue27–30 or a radical. Subsequent electron donation from the PS radiby using two-photon excitation where two NIR photons cal anion to oxygen produces the superoxide anion radical are simultaneously delivered in a very short pulse to be (O−• 2 ). Therefore, the efficient ISC process giving a high equivalent to one photon of twice the energy and shorter quantum yield of triplet state is essential for the generation wavelength.31–35 of ROS. Strong absorbtion of light, high triplet state quantum yield (effective ISC) and a long-lived triplet excited Photophysics and Photochemistry of state are required to be an ideal PS. However, there has Fullerenes and Derivatives not yet been established a clear relationship between the Fullerenes with attached side chains, called "functionalefficiency of ISC and the chemical structure. ized fullerenes," are known to demonstrate high efficiency in the formation of singlet oxygen, hydroxyl radicals and FULLERENES AS PHOTOSENSITIZERS superoxide anions, which are considered as effective mediBesides occupying an important place in biomedicine, ators of PDT. The photophysics of fullerenes is quite nanoparticles have also been shown to have potential to favorable for PDT. The absorption spectra of a typical act as a photosensitizing drug in PDT. Fullerenes are conset of mono-substituted, bis-substituted and tris-substituted sidered to have advantages as potential PS on the basis fullerenes show almost monotonic decay between 300 and of certain favorable PDT characteristics when compared 700 nm.20 When C60 is irradiated with visible light, it is 19 to conventional PS with the tetrapyrrole structure. The excited from the S0 ground state to a short-lived (< 1.3 ns) fullerenes are known for their photostability, a prerequiS1 excited state. The S1 state quickly decays to triplet site to behave as an effective PS, and they undergo relstate. The triplet yield is 1 and the lifetime is as long atively less photobleaching than the tetrapyrroles. They as 50–100 s. By energy transfer mechanism (Type II) can also be modified to get the desired the degree there is the generation of singlet oxygen (1 O2 ) by quench20 lipophilicity by chemical functionalization. There is ing of fullerenyl T1 state in the presence of dissolved J. Biomed. Nanotechnol. 10, 1918–1936, 2014
Schematic of PDT mediated by fullerenes.
molecular oxygen. At 532 nm excitation the singlet oxydown or trapped for a sufficient time allowing the two gen quantum yield ( ) for this process is close to theoradicals to react with each other, which produce the comretical maximum, 1.0.36 Calculations in 1986 by Haddon paratively less-reactive ROS, hydrogen peroxide. However, et al. had indicated that the lowest unoccupied molecular the mechanism may be significantly different with more orbital (LUMO) of C60 would be low-lying, triply degenwater-compatible C60 derivatives.45 37 erate, and, hence, capable of accepting up to 6 electrons. The pristine C60 itself alone is not a good PS due to the DESIGN OF FULLERENE DERIVATIVES very weak absorption of visible light and water insoluDelivered by Ingenta to: University Carolina One of of theSouth concerns that have been raised about the use bility. However, C60 is an ideal spin converter due to its IP: 46.161.56.113 On: Mon,of23fullerenes May 2016concerns 08:28:44their biodegradability, as nanostate energy level Scientific Publishers efficient inherent ISC and a low S1Copyright: American structures have the possibility of accumulation in the body (about 1.72 eV). When a visible-light-harvesting antenna during blood circulation or in the environment after use.46 is attached onto C60 to produce C60 -dyads, it turns out to 38 Though it has been shown that pristine C60 is nontoxic be a potentially ideal PS. Figure 1 shows a schematic its insolubility and its pronounced tendency to aggreoutline centered upon a Jablonski diagram of PDT medigate decreases its potential to be useful in biomedicine. ated by fullerene and its derivatives. Many strategies have been demonstrated or applied to Both pristine and functionalized fullerenes have the 39 either solubilize or modify fullerenes for improving potential to produce ROS after illumination. The ROS their utility in drug-delivery and medical applications. produced by fullerene derivatives during illumination are The following approaches: liposomes;47–49 micelles;50 51 inclined towards Type I photochemical products (superdendrimers;52 53 PEGylation;53–56 self-nanoemulsifying oxide radical, hydroxyl radical, lipid hydroperoxides, and systems (SNES);57–60 encapsulation in cyclodextrins.47 61 62 hydrogen peroxide), compared to the standard Type II ROS have all been explored by various groups throughout the (singlet oxygen) for most PS. During this process they are world to overcome this problem with fullerenes. able accept electrons very efficiently as many as six to 40 41 It is known that cationic functional groups provide each C60 cage. It is thought that the reduced fullerene good solubility for molecules of diverse structures and triplet or radical anion can transfer an electron to molecalso have the potential to bind to the anionic residues ular oxygen, forming the superoxide anion radical.42 One present on cancer cells and also on the bacterial cell wall apparent incongruity that arises in this area needs to be via static charge interactions. For these reasons cationic addressed. It is a well-known fact that fullerenes can act groups can be considered a good choice for attachment as antioxidants, and that C60 and derivatives can act as on the fullerene cage. A number of chemical functionscavengers of ROS in the absence of light. One clue that alization techniques for derivatizing fullerenes have been might explain this inconsistency was proposed in 2009, by evaluated.63 64 Among them, general suitable methods for Andrievsky et al.43 who showed that the major mechanism the preparation of cationic fullerene derivatives include by which hydrated C60 can inactivate the highly reactive − cyclopropanation65 e.g., C60 [> M(C3 N+ ROS, hydroxyl radical, not by covalently scavenging the 6 C3 )2 ] − (I )10 + 66 67 − (LC14, Fig. 2(a)) and C70 [> M(C3 N6 C3 )2 ] − (I )10 radicals but rather by action of the coat of "ordered water" (LC17, Fig. 2(b))68 and pyrrolidination e.g., quaternized that was linked with the fullerene nanoparticle.44 One of dimethylpyrrolidinium fullerenyl monoadduct (BF4,20 the explanations is that hydroxyl radicals can be slowed 1922
The chemical structure of fullerene derivatives applied in PDT studies.
Fig. 2(h)) is also a light-harvesting donor chromophore Fig. 2(c)) and trisadduct (BF6,69 Fig. 2(d)) and structures − antenna that can be attached to the C60 cage to faciliC ) ]−(I ) , and LC24, such as LC22, C60 [> DPAF(MN+ 3 2 10 6 − 70 71 tate ultrafast intramolecular energy- and electron-transfer C70 [> M(C3 N+ 6 C3 )2 ]2 − (I )20 . Delivered Ingenta to: University Southantenna Carolina from theofdonor to C60 and can therefore be used The cyclopropanation reaction of C60 by functionalizaIP: 46.161.56.113 On: Mon, 23 May 2016 08:28:44 to enhance PDT efficacy.73 DPAF-C2M was constructed to tion was recently applied to attach a highly complex Copyright: American Scientific Publishers have increased optical absorption at 400 nm and also posdecacationic moiety to the fullerene cage leading to sessed good two-photon absorption (2PA) cross-sections the formation of fullerenes bearing ten positive charges. in the NIR wavelengths. Later another set derivatives The decacationic functional moieties of C60 , C70 , and C60 (> CPAF-C2M (Fig. 2(i)) was formed by structural C84 O2 fullerenes were designed to increase both the modification via chemical conversion of the keto group water-solubility and provide surface binding interactions in C60 (> DPAF-C2M ) to a stronger electron-withdrawing with –D-Ala-D-Ala residues of the bacteria cell wall by 1,1-dicyanoethylenyl (DCE) unit. This structural modifiincorporating multiple H-bonding interactions and poscation induced a large bathochromic shift of ground-state itive quaternary ammonium charge to bind to anionic absorption of CPAF-C2M moieties beyond 450–550 nm lipopolysaccharides and lipoteichoic acids.70 The strucand an increased electronic polarization of the molecule. ture included two esters and two amide moieties to give The modification also led to a large bathochromic shift a sufficient number of carbonyl and –NH groups in a of the major band in visible spectrum giving measureable short length of ∼ 20 Å to provide effective multi-binding absorption up to 600 nm and extended the photoresponsive sites with the presence of a well-defined water-soluble capability of C60 –DCE–DPAF nanostructures to longer red C -OH at each side of the pentacationic moiety C3 N+ 6 3 wavelengths than C60 (> DPAF-C2M ). arm. A similar reaction sequence with a malonate preIt was reported that the majority of the HOMO eleccursor arm M(C3 N6 C3 )2 was also employed in the prepa− tron density was delocalized over the dialkyldiphenyC ) ][> M(C N C ) ] − (I ) ration of C70 [> M(C3 N+ 3 6 3 2 10 6 3 2 ↓ laminofluorene (DPAF-Cn ) moiety, whereas the LUMO (LC18, Fig. 2(e)), C84 O2 [> M(C3 N+ C ) ] − (I ) (LC19, 10 6 3 2 − electron density was located on the C60 spheroid.74 C ) ][> M(C N C ) ] − (I ) Fig. 2(f)), C84 O2 [> M(C3 N+ 3 6 3 2 10 6 3 2 67 Therefore charge-separated states may be generated by (LC20, Fig. 2(g)). intramolecular electron-transfer between the diphenyTo circumvent the shortcoming of weak absorption of laminofluorene donor and C60 > acceptor moieties during light at visible wavelengths, a variety of highly fluoresthe photoexcitation process. cent donor chromophore antennae have been covalently Intramolecular formation of transient charge-separated attached to the fullerene, such as porphyrins.72 Fullerenestates is crucial for generation of radical ROS, initially porphyrin hybrids are more efficient in terms of singlet • −• +• with O−• oxygen generation and also have improved cell penetra2 and subsequently HO . C60 (> CPAF -Cn ) is the most stable charge-separated state in polar solvents, tion. Dialkyldiphenylaminofluorene (DPAF-C2M , shown in J. Biomed. Nanotechnol. 10, 1918–1936, 2014
including H2 O.74 In nonpolar solvents, the intramolecular energy-transfer event that produces 3 C∗60 (> CPAF-Cn ) transient state dominates with nearly 6-fold higher generation of singlet O2 compared to 3 C∗60 (> DPAF-Cn ).
methods of fullerenes have been reviewed.64 76–78 Among them, common convenient methods for the preparation of cationic fullerene derivatives include cyclopropanation79 and pyrrolidination80 reactions due to their high consistency allowing product reproducibility. Examples of the latter were given in the preparation of quaternized dimethylpyrrolidinium [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 -methylglycine (sarcosine) and paraformalaldehyde in toluene at the reflexing temperature to afford either mono-N -methylpyrrolidino[60] fullerene (BF4) or a large number of regioisomers of tris(N -methylpyrrolidino)[60] fullerene (BF6) derivatives, as shown in Figure 3. Upon quaternization of these intermediates using methyl iodide as the methylation agent, corresponding monocationic and tricationic products as BF4 and BF6 were obtained, respectively.
Examples of the Synthesis of Monocationic and Polycationic Fullerene Derivative As mentioned above cationic functional groups are generally considered as the addend of choice for attachment on the fullerene cage due to their potential surface binding contact with anionic residues of the bacteria cell wall via static charge interactions. A systematic trend to increase the number of positive charges per fullerene cage was described in a recent report75 to maximize such interactions and use them as the approach for targeting bacteria having a significant density of anionic residues at the cell wall surface. A number of chemical functionalization
H2C=O
reflux toluene
(1.0 or 3.0 equiv.)
N CH3I
CH3I
Delivered by Ingenta to: University of South Carolina IP: 46.161.56.113 On: Mon, 23N May 2016 08:28:44 N Copyright: American Scientific Publishers
BF6 H
+ N
O OH
H (1.0 or 2.0 equiv.)
CH=O or
N N CH3I CH3I N
BF22 BF24
Figure 3. Synthesis of monocationic and tricationic dimethylpyrrolidinium [60]fullerenes tively, and mono- and bis(piperazinopyrrolidinium) [60]fullerenes BF22 and BF24, respectively.
BF6,
respec-
H2N
N6C3
C70[>M(t-Bu)2] C3N6C3-OH
(CF3CO2 )5
(CF3CO2 )5 C70[>M(C3N6+C3H)2]
C70[>M(C3N6+C3)2] I
Delivered by Ingenta to: University of South Carolina IP: 46.161.56.113 On: Mon, 23 May 2016 08:28:44 Copyright: American Scientific Publishers C60[>M(C3N6+C3)2] Figure 4.
Synthetic scheme for the preparation of C60 [> M(C3 N+6 C3 )2 ] and C70 [> M(C3 N+6 C3 )2 ].
Similarly, the reaction of C60 in toluene with either 1.0 or 2.0 equivalent of azomethine ylide produced by piperazine-2-carboxylic acid dihydrochloride dissolved in methanol and triethylamine in the presence of 4-pyridinecarboxaldehyde at the refluxing temperature gave the corresponding mono-piperazinopyrrolidino[60] fullerene or a number of regioisomers of bis(piperazinopyrrolidino)[60] fullerene derivatives. Quaternization of both intermediates with methyl iodide led to the corresponding monocationic and dicationic products as BF22 and BF24 (Fig. 3), respectively. In the case of cyclopropanation reaction of C60 as the functionalization method, it was applied recently for the attachment of a highly complex decacationic moiety to the fullerene cage leading to the formation of + 83 In this C60 [> M(C3 N+ 6 C3 )2 ] and C70 [> M(C3 N6 C3 )2 ]. reaction, a malonate precursor arm was applied to include two esters and two amide moieties, for a sufficient number of carbonyl and –NH groups in a short length of ∼ 20 Å, with a well-defined water-soluble pentacationic moiety N+ 6 C3 at each side of the arm for making effective multi-binding sites to the cell wall. The precursor N+ 6 C3 was reported to be a common synthon J. Biomed. Nanotechnol. 10, 1918–1936, 2014
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 N6 C3 . The best method for the preparation of C60 [> M(C3 N+ 6 C 3 )2 ] and C70 [> M(C3 N+ 6 C3 )2 ] was depicted in Figure 4 to begin with a well-defined fullerene monoadduct derivatives, such as di(tert-butyl)fullerenyl malonates C60 [> M(t-Bu)2 ] and C70 [> M(t-Bu)2 ], respectively, followed by facile transesterification reaction with the well-characterized tertiary-amine precursor arm moiety, 4-hydroxy-[N ,N ',N ,N ,N ,N -hexapropyl-hexa(aminoethyl) butanamide (C3 N6 C3 -OH) using trifluoroacetic acid as the catalytic reagent to afford protonated quaternary ammonium trifluoroacetate salt C70 [> M(C3 N+ 6 C3 H)2 ]. C Conversion of this salt to C70 [> M(C3 N+ 6 3 )2 ], was accompanied by neutralization of trifluoroacetic acid by sodium carbonate and subsequent quaternization by methyl iodide to give decacationic quaternary ammonium iodide salts. A similar conversion procedure was applied for the case of C60 [> M(C3 N+ 6 C3 )2 ]. This synthesis represented the first examples of decacationic fullerene monoadducts to incorporate a well-defined high number of cation without the use of multiple addend attachments to 1925
Figure 5. (FC4 S).
Synthesis of hexaanionic hexa(sulfo-n-butyl)-C60
Figure 6. (A) Structure of hexa(sulfo-n-butyl) [60]fullerene (FC4 S) and (B) a characterized FC4 S-derived nanosphere formed in H2 O discussed by Yu.85
the light-harvesting donor chromophore antenna attachment at a very close vicinity of C60 cage, within a contact distance of 2.6–3.5 Å, to facilitate ultrafast intramolecular energy- and electron-transfer from the donor antenna Synthesis of Hexa-Anionic Fullerene Derivatives to C60 for enhancing PDT efficacy.73 A specific donor Fullerene molecules are highly hydrophobic. Pristine C60 antenna, namely, dialkyldiphenylaminofluorene DPAF-Cn can be dispersed into aqueous medium in a micelle form was first introduced to give an increased optical absorpwith the application of surfactants. However, the micelle tion at 400 nm and later being modified by replacing structure may not be stable enough in biological environthe keto moiety of DPAF-Cn via a highly electronment. In a recent report, a strategy of creating a micelle withdrawing 1,1-dicyanoethylenyl (DCE) bridging group formed from surfactants covalently bonded directly onto that resulted in dark burgundy-red C60 (> CPAF-Cn ) derivathe fullerene cage was illustrated by the synthesis of tives. This structural modification was found to induce hexa(sulfo-n-butyl)-C60 (FC4 S) leading to structurally staa large bathochromic shift of ground-state absorption of ble molecular micelles in H2 O.84 The synthesis involved CPAF-Cn moieties beyond 450–550 nm. 6 the use of hexaanionic C60 (C60− ) chemistry85 for attachPreparation of C60 (> CPAF-C2M , as an example, ing six sulfo-n-butyl arms on CDelivered reaction,to: as University 60 in one-pot was made by Friedel-Craft by Ingenta of South Carolina acylation of 9,9-dimethoxyshown in Figure 5. IP: 46.161.56.113 On: Mon,ethyl-2-diphenylaminofluorene 23 May 2016 08:28:44 with bromoacetyl bromide Copyright: of American It was found that molecular self-assemblies FC4 S Scientific in the Publishers presence of AlCl3 to yield 7-bromoactylresulted in the formation of nearly monodisperse 9,9-dimethoxyethyl-2-diphenylaminofluorene, followed by spheroidal nanospheres with the sphere radius of gyration cyclopropanation reaction with C60 , as shown in Figure 7. Rg ≈ 19 Å, where the major axe ≈ 29 Å and the minor axe The resulting product C60 (> DPAF-C2M ) was then further ≈ 21 Å for the ellipsoid-like aggregates, or an estimated treated with malononitrile and pyridine in the presence of long sphere diameter of 60 Å [the radius = (5/3)1/2 Rg ] titanium tetrachloride in dry toluene to yield C60 (> CPAFfor the aggregates, as determined by small angle neutron C2M ) after chromatographic purification. scattering (SANS) in D2 O and small angle X-ray scattering (SAXS) in H2 O.86 This radius of gyration was found Photochemistry and Photophysics of Fullerenyl to remain relatively constant over a concentration range Molecular Micelles and Chromophore-Fullerene from 0.35 to 26 mM in H2 O, revealed strong hydrophobic Conjugates interaction between core fullerene cages overcoming loose Photoexcitation of C60 and fullerene derivatives induces a charge repulsion at the surface of the molecular micelle. singlet fullerenyl excited state that is transformed to the It allowed the nanosphere formation at a low concentration despite of steric hindrance and high hydrophilicity arising from of 6 sulfo-n-butyl arms surrounding C60 . Based on the SANS data, the mean number of FC4 S molecules for the nanosphere was determined to be 6 5 ± 0 7 that led to the elucidation of its nanocluster structure with each FC4 S molecule located at the vertex of an octahedron shaped nanosphere shown in Figure 6.85 preserve the intrinsic photophysical properties of fullerene cages.
Synthesis of Chromophore-Linked Fullerene Derivatives Optical absorption of C60 is strong in UVA and weak in most of visible range. To circumvent this shortcoming, a reported approach was described to use 1926
Figure 7. Synthetic method of C60 (> CPAF-C2M ) discussed by Chiang.73 J. Biomed. Nanotechnol. 10, 1918–1936, 2014
corresponding triplet excited state, via intersystem energy non-polar solvents, observed short fluorescence lifetime of the model compound 1 CPAF∗ -C9 (241 ps) as compared crossing, with nearly quantitative efficiency.41 Subsequent with that of the keto analogous Br-1 DPAF∗ -C9 (2125 ps) energy transfer from the triplet fullerene derivatives to may be indicative of a facile photoinduced intramolecumolecular oxygen produces singlet molecular oxygen in lar charge polarization process forming the correspondaerobic media. This photocatalytic effect becomes one ing [C C(CN)2 ]−• –DPAF+• -C9 charge-separated state that of key mechanisms in photodynamic treatments using +• will facilitate the formation of C−• fullerene derivatives as photosensitizers. However, a high 60 (> CPAF -Cn ) in the subsequent electron-transfer event. degree of functionalizaton on C60 for the enhancement of solubility and compatibility in biomedia resulted in a progressive decrease of the singlet oxygen producANTICANCER EFFECT OF FULLERENE-PDT tion quantum yield [ 1 O2 )]. Examples were given by There have been various studies demonstrating fullerene Bingel-type malonic acid, C60 [C(COOH)2 ]n , and malonic induced in-vitro phototoxicity in cells. It is considered ester, C60 [C(COOEt)2 ]n , [60] fullerene adducts,87 showing that one condition for any PS to produce cell killing a decreasing trend of 1 O2 ) as the number of addends (n) after illumination, is that the PS should really be taken increases. When the number n reached 6 for a hexaadduct, up inside the cell, as the production of ROS outside the its (1 O2 ) value declined to only 13% or less of that for cell will not be enough to produce cell death unless it is C60 .88 However, it was not the case for molecular micellar produced in extremely large amounts. One of the limFC4 S, a relatively high singlet oxygen production quanitations for the study of the uptake of fullerenes into tum yield for FC4 S may indicate its unique electronic feacells is their non-fluorescent nature that limits the use tures in difference with Bingel-type malonic hexaadducts of fluorescence microscopy to study the localization in of C60 .85 The efficiency was substantiated by direct deteccells. Some strategies though have been adopted to overtion of 1 O2 emission at 1270 nm upon photoirradiation of come this limitation, such as the use of radiolabeled self-assembled FC4 S nanospheres at 500–600 nm. fullerene that has been prepared to study the uptake. In the cases of light-harvesting electron-donor chroIndirect immunofluorescence staining with antibodies has mophore assisted fullerene conjugate systems, such been used to show the localization of fullerene in mitoas C60 (> CPAF-Cn ) derivatives, their photophysical chondria and other intracellular membranes.89 Recently properties involve the primaryDelivered photoexcitation events energy-filtered transmission by Ingenta to: University of South Carolina electron microscopy and elecof either the fullerene moiety IP: at 46.161.56.113 UV wavelengths On:orMon,tron 23 tomography May 2016 08:28:44 was used to visualize the cellular uptake the DPAF-Cn moiety at both UV Copyright: and visibleAmerican wave- Scientific of pristinePublishers C60 nanoparticulate clusters in the plasma memlengths up to 600 nm.73 Much higher optical absorption brane, lysosomes and in the nucleus of cells.90 capability of DPAF-Cn than the C60 > cage in visible wavelengths enables the former moiety to serve as a lightIn Vitro Anti-Cancer PDT with Fullerenes harvesting antenna. Accordingly, formation of the phoThe first report of phototoxicity in cancer cells meditoexcited 1 (DPAF)∗ -Cn moiety should be considered as the ated by fullerenes was in the year 1993. In this study early event in the photophysical process. Alteration of the Tokuyama et al.91 used carboxylic acid functionalized keto group of C60 (> DPAF-Cn ) to the 1,1-dicyanoethylenyl fullerenes at 6.0 M and white light to produce growth group of C60 (> CPAF-Cn ) effectively extended its photoreinhibition in cancer cells. Burlaka et al.92 used pristine sponsive region to longer red wavelengths. Photoexcitation C60 at 10 M with visible light from a mercury lamp processes of C60 (> DPAF-Cn ) and C60 (> CPAF-Cn ) pump to produce some phototoxicity in Ehrlich carcinoma cells an electron from their highest occupied molecular orbital or in rat thymocytes. The cytotoxic and photocytotoxic (HOMO) to the lowest unoccupied molecular orbital effects of two water-soluble fullerene derivatives, a den(LUMO). By the molecular orbital calculation and energy dritic C60 monoadduct and the malonic acid C60 trisadduct minimization, the majority of the HOMO electron density were tested on Jurkat cells when irradiated with UVA or was reported to be delocalized over the dialkyldiphenyUVB light.93 The cell death was mainly caused by memlaminofluorene (DPAF-Cn ) moiety, whereas the LUMO brane damage and it was UV dose-dependent. electron density was located on the C60 spheroid, and New approaches have been tested to overcome the +• therefore C−• requirement to utilize UV or short-wavelength visible 60 (> CPAF -Cn ) was suggested as the most stable charge-separated (CS) state in polar solvents, includlight to activate fullerenes. In one study where two new ing H2 O.74 These charge-separated states may be generated fullerene-bis-pyropheophorbide-a derivatives were preby photoinduced intramolecular electron-transfer between pared: a mono-(FP1) and a hexaadduct (FHP1). The C60 the diphenylaminofluorene donor and C60 > acceptor moihexaadduct FHP1 had a significant phototoxic activity eties. The process effectively quenches fluorenyl fluores(58% cell death, after a dose of 400 mJ/cm2 of 688 nm cence that can be observed in the most of C60 (> DPAF-Cn ) light) but the monoadduct FP1 had a very low photoand C60 (> CPAF-Cn ) monoadducts. Even during energytoxicity and only at higher light doses.94 Nevertheless transfer events of C60 (> CPAF-Cn ), normally favorable in the activity of both adducts was less than that of pure J. Biomed. Nanotechnol. 10, 1918–1936, 2014
− pyropheophorbide-a, possibly due to the lower cellular (LC19) and C84 O2 [> M(C3 N+ 6 C3 )2 ][> M(C3 N6 C3 )2 ]-(I )10 95 67 uptake of the adducts. (LC20) (shown in Figs. 2(f) and (g)). Different waveThe hypothesis that fullerenes have the potential to lengths were used for irradiation. UVA and blue light caused more killing with LC20 than with LC19. This difdestroy cancer cells by PDT was tested in our group. We ference can be attributed to better chance of electron transhave shown that the C60 molecule mono-substituted with a fer process occurring with shorter wavelengths and also single pyrrolidinium group (BF4 shown in Fig. 2(c)) is an the presence of the electron donating tertiary-polyethyleneefficient PS and can mediate killing of a panel of mouse amine chain. While when white light was employed the cancer cells.20 The cells lines used were lung cancer (LLC) variation between LC20 and LC19 was smaller but LC20 and colon cancer (CT26) adenocarcinoma and reticulum still gave extra killing, while green light gave equivalent cell sarcoma (J774) and the latter showed much higher suskilling for the two fullerenes. The situation was upturned ceptibility to fullerene mediated phototoxicity possibly due and LC19 gave considerably more killing than LC20 when to having an increased uptake because J774 cells behave red light was used. It is important to state that the comlike macrophages. Besides the exceptionally active BF4, pounds used here induced a very low dark toxicity. the next group of compounds has only moderate activity (BF2, BF5, and BF6 shown in Fig. 2(d)) against J774 cells showing only some killing even at high fluences, while In Vivo Photodynamic Therapy of Cancer last two compounds (BF1 and BF3) had no detectable The three prerequisites for the fullerene PS to have phoPDT killing up to 80 J/cm2 . The indirect measurement todynamic effect on tumors are first of all it should accuof fullerenes uptake was demonstrated by increase in flumulate in the tumor tissue; secondly there should be a orescence of an intracellular probe (H2 DCFDA) which practically efficient way to administer the compound to is specific for the formation of ROS. We also showed tumor bearing animals; and thirdly a practical way to the initiation of apoptosis by PDT mediated by BF4 deliver excitation light to the tumors.100 The first chaland BF6 in CT26 cells at 4−6 h after illumination. lenge in this direction was taken up by Tabata56 in 1997. The induction of apoptosis was rapid after illumination To make the water-insoluble pristine C60 water soluble which may perhaps suggest that fullerenes are localized and enlarge its molecular size they chemically modified in mitochondria, as it has been previously shown with it with polyethylene glycol. This conjugate was injected benzoporphyrin derivative.96–98 Delivered The explanation for to: the University intravenously intoCarolina mice carrying a subcutaneous tumor on by Ingenta of South mono-pyrrolidinium substituted IP: fullerene as most effecconjugate demonstrated higher accumu46.161.56.113 On: Mon,the 23back. May C 2016 08:28:44 60 -PEG Copyright: American tive PS most likely linked to its relative hydrophobicity as Scientific lation andPublishers relatively more prolonged retention in the tumor established by its log P value of over 2. Besides this a sintissue than in normal tissue. On performing PDT after gle cationic charge on BF4 is in addition expected to play intravenous injection of C60 -PEG conjugate or Photofrin to a significant role in determining its relative phototoxicity. tumor-bearing mice, coupled with exposure of the tumor Elisa Milanesio et al.97 used tetrapyrrole-fullerene consite to visible light, the volume increase of the tumor mass jugates and evaluated PDT effect with a porphyrin-C60 was suppressed and the C60 _PEG conjugate exhibited a dyad (P-C60 ) and its metal complex with Zn(II) (ZnP-C60 stronger suppressive effect than Photofrin. Tumor necrosis and compared with 5-(4-acetamidophenyl)-10,15,20-tris(4was observed without any damage to the overlying normethoxyphenyl)porphyrin (P) on Hep-2 human larynx carmal skin. The antitumor effect of the conjugate showed an cinoma cell line. The phototoxicity was dependent on light increase with increasing fluence delivered and C60 dose, exposure level with visible light. 80% phototoxicity was and cures were achieved by treatment with a low dose of observed for P-C60 after 15 min of light irradiation which 424 g/kg at a fluence of 107 J/cm2 . In another study was higher as compared to ZnP-C60 . In case of argon atmoLiu and others55 conjugated polyethylene glycol (PEG) sphere also a high photoactivity was observed with both to C60 (C60 -PEG), and diethylenetriaminepentaacetic acid the dyads. In another paper,99 the cell death was confirmed (DTPA) was subsequently introduced to the terminal group to occur by apoptotic mode. of PEG to prepare C60 -PEG-DTPA that was mixed with As fullerenes show a relatively slower uptake we incugadolinium acetate solution to obtain Gd3+ -chelated C60 + − bated the cells for 24h with C60 [> M(C3 N6 C3 )2 ]-(I )10 PEG-DTPA-Gd. PDT induced anti-tumor effect and MRI − (LC14) and C70 [> M(C3 N+ tumor imaging was evaluated on intravenous injection of 6 C3 )2 ]-(I )10 (LC17) (shown − in Figs. 2(a) and (b)). C70 [> M(C3 N+ C60 -PEG-DTPA-Gd into the tumor bearing mice. Equiv6 C3 )2 ]-(I )10 killed − cells more effectively than C60 [> M(C3 N+ alent generation of superoxide upon illumination was 6 C3 )2 ]-(I )10 , On the contrary, the fullerene drug LC14 killed less than observed with or without Gd3+ chelation. Intravenous 1 log at all fluencies. LC17 that was short of the decainjection of C60 -PEG-DTPA-Gd into tumor bearing mice tertiary amine chain was less phototoxic than LC18 which plus light (400∼500 nm, 53.5 J/cm2 ) demonstrated sigpossessed an extra deca-tertiary ethyleneamine chain. nificant anti-tumor PDT depending on the timing of light This exciting result prompted us to carry out studies irradiation that also correlated with tumor accumulation as − detected by the enhanced MRI signal. with new PDT compounds C84 O2 [> M(C3 N+ 6 C3 )2 ]-(I )10
Figure 8. (A) Bioluminescence imaging of CT26-Luc tumors growing in a representative control mouse (upper panel) and a representative IPPDT treated mouse (lower panel). (B) Quantitative analysis of bioluminescence dynamics in control and white light treated mice (n = 10 per group). Reprinted with permission from [102], P. Mroz, et al., Intraperitoneal photodynamic therapy mediated by a fullerene in a mouse model of abdominal dissemination of colon adenocarcinoma. Nanomedicine 7, 965 (2011). © 2011, Future Science.
be more soluble. These conjugates demonstrated selective Yu and her coworkers101 performed a preliminary in vivo study of PDT using hydrophilic nanospheres formed from phototoxicity compared to fibroblast cells thus suggesthexa(sulfo-n-butyl)-C60 (FC4 S, shown in Fig. 3(A)). This ing the significance of targeting glucose receptors.103 The study was performed on ICR mice bearing sarcoma PDT effect in vivo was investigated in human-melanoma 180 subcutaneous tumors. No adverse effects were noted (COLO679)-xenograft bearing mice by injecting C60 in the animals when the FC4 S was administered orally. (Glc)1 (0.1 or 0.2 mg/tumor) intratumorally followed by Water-soluble FC4 S in PBS (5 mg/kg body weight) was irradiated with 10 J/cm2 UVA 1ight. The drug-light intergiven either intraperitoneally or intravenously with subseval was 4 h. Tumor growth was suppressed better with the quent irradiation with an argon ion laser beam at a wavehigher dose than the lower dose. Delivered by Ingenta to: University of South Carolina length of 515 nm or an argon-pumped dye-laser at 633 nm. IP: 46.161.56.113 The beam was focused to a diameter of 7–8 mm withOn: theMon, 23 May 2016 08:28:44 Copyright: American Scientific Publishers EFFECT OF FULLERENES ANTIMICROBIAL 2 total light dose of 100 J/cm . Inhibition of tumor growth Antibiotic resistance is a worldwide problem that is was found more effective using the low wavelength i.e., in spreading with remarkable speed. The injudicious and case of better-absorbed 515nm laser than the 633 nm laser. over-use of antibiotics is the most important reason leading I · p. administration method proved to be slightly better in to antibiotic resistance around the world. inhibition effectiveness than the i.v. method. Conclusively The "golden age" of antimicrobial therapy began with data suggest that PDT with fullerenes is not only possible the discovery of antibiotics around the middle of the in animal tumor models, but can demonstrate the potential last century.104 Meanwhile many other ancient effective use of these compounds as PS for PDT of cancer. 102 antibacterial treatments including photosensitizing reacWe have also recently shown the therapeutic effects tions were forgotten. In the last few decades, however, of intraperitoneal PDT with fullerene and white light in the widespread use of antimicrobial agents emerges the a very challenging mouse model of disseminated abdomincrease of antibiotic-resistant bacteria and other infectious inal cancer. In this study we prepared the monocationic microorganisms and led to predictions of untreatable infecBF4 (Fig. 2(c)) in micelles composed of Cremophor EL. tions caused by "superbugs,"105 which in turn has created Colon adenocarcinoma cell line (CT26) expressing firean ever-increasing need for new drugs. fly luciferase was used to allow monitoring of IP tumor Therefore antimicrobial PDT has become an emerging burden by non-invasive biolumi8nescence imaging. BF4 in alternative strategies for destroying microorganisms espemicelles was injected intraperitonally (5 mg/kg) followed cially for multi-drug resistant pathogens.106 PDT produces by white-light illumination (100 J/cm2 ) delivered through ROS that are toxic to the target microorganisms. PDT has the peritoneal wall. This produced a statistically significant a broad spectrum of action, and compared to antibiotic reduction in bioluminescence and besides this produced a treatment PDT does not lead to the selection of mutant survival advantage in mice, shown in Figure 8. A drugresistant strains. light interval of 24 h was more effective than a 3 h drugCurrently, topical application of a PS on infected tislight interval showing the significance of allowing enough sues and subsequent illumination seems to be the most time for the fullerene to be taken up into the cancer cells. prominent feature of antimicrobial PDT, without damaging As the cancer cells are known to express more glucose the surrounding tissue or disturbing the residual bacterialreceptors, Otake et al. exploited this fact and synthesized group of C60 -glucose conjugates which also proved to flora. It was well accepted that Gram-positive bacteria are J. Biomed. Nanotechnol. 10, 1918–1936, 2014
more susceptible to PDT as compared to Gram-negative bacteria. This can be explained by the different structures of their cell walls.107 There are several possible mechanisms to explain the antimicrobial activity of illuminated fullerene PS: by interfering with cell wall synthesis; plasma membrane integrity; nucleic acid synthesis; ribosomal function and folate synthesis. All of these would result in disruption of the bacterial cell function and inhibit their growth. Martin and Logsdon hypothesized that it was possible that microorganisms were susceptible to damage by to Type I ROS when compared with Type II singlet oxygen.108 As mentioned before fullerenes can gain solubility63 and produce more hydroxyl radicals and superoxide anion, as well as singlet oxygen through functionalization109 by attaching some hydrophilic or amphiphilic functional groups.22 An ideal PS proposed for antimicrobial PDT can be judged on several criteria. These PS should have no toxicity in the dark and should selectively kill bacteria over mammalian cells. PS should be able to kill multiple classes of microorganisms at relatively low concentrations with low fluences of light. PS should ideally have high absorption around 600 nm to 800 nm and generate high triplet and singlet oxygen quantum yields.
more traditional PS such as tetrapyrroles that generate singlet oxygen. These compounds all performed significantly better 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 derivative was an interesting PS with potential applications in PDI of bacteria. They compared the PDI efficacy of fullerene derivatices with different numbers of cationic charges. The spectroscopic and photodynamic properties of a dicationic N N -dimethyl-2-(40-N ,N ,N -trimethylaminophenyl) fulleropyrrolidinium iodide) (DTC2+ 60 ) were compared with a non-charged N -methyl-2-(4 -acetamidophenyl)fulleropyrrolidine (MAC60 ) and a monocationic N ,N -dimethyl-2-(4 -acetamidophenyl)fulleropyrrolidinium iodide (DAC+ 60 ) in different media and in a typical Gram-negative bacterium, E. coli. PDI of E. coli cellular suspensions by dicationic fullerene exhibits a ∼ 3.5 log decrease of cell survival after 30 min of irradiation, which represents about 99.97% of cellular inactivation. To determine the optimal chemical structures produced by fullerene derivatization, a QSAR relationship study employed fullerene PS with a wider range of different hydrophobicities, as well as with an increased number of cationic charges.113 The results indicated that increasing the number of cationic charges and lowering the hydrophobicAntimicrobial Effects In Vitro ity tended to increases the antimicrobial PDI efficacy of Delivered by Ingenta to: University of South Carolina fullerene PS against both The structures, especially the charges of attached groups IP: 46.161.56.113 On: Mon, 23 May 2016 08:28:44Gram-positive and Gram-negative Copyright: American bacteria. Publishers The charge increases the association of the PS on fullerene influence the efficacy of PS on killing Scientific with negatively charged pathogen membranes, whereas the microorganisms. Our lab has shown, in a series of reported hydrophobic character increases association with or penexperiments that cationic fullerenes fulfill many, but not etration into the lipid components of the membrane, or all of the aforementioned criteria. At the first time we both. Recently, Mizuno et al.82 from our laboratory emphademonstrated that the soluble functionalized fullerenes sized the importance of the number of cationic charges in were efficient antimicrobial PS and could mediate selecinfluencing the efficiency of the fullerenes in antimicrobial tive photodynamic inactivation (PDI) for various classes 75 PDI when they looked at a further series of functionalized of microbial cells over mammalian cells. We compared cationic fullerenes PS. They compared PDI efficacy of a the antimicrobial activity of broad-spectrum antimicrobial new group of synthetic fullerene derivatives that possessed photodynamic activities of two series of functionalized either basic or quaternary amino groups as antimicrobial PS C60 ; a first series with one, two, or three polar diseriagainst S. aureus (Gram-positive), E. coli (Gram-negative) nol groups, and a second series with one, two, or three bacteria and C. albicans (fungi). QSAR derived with Log P quarternary pyrrolidinium groups. Gram-positive bacteria and hydrophilic lipophilic balance parameters showed that (S. aureus) Gram-negative bacteria (E. coli and P. aerugmuch better correlations were obtained when 3× the numinosa), and fungal yeast (C. albicans) were tested in this ber of cationic charges were subtracted from the Log P valstudy. The neutral, alcohol-functionalized fullerenes had ues. The most effective ones to perform antimicrobial PDT only modest activity against S. aureus, while the cationic were tetracationic compound BF21 that had more cationic pyrrolidinium-functionalized fullerene (BF6, Fig. 2(d)) charges and a lower log P. S. aureus was most susceptible; was surprisingly effective in causing light-mediated killing E. coli was intermediate, while C. albicans was the most of S. aureus at 1 M, and 10 M for E. coli, C. albicans resistant species tested. and P. aeruginosa. However, the pyrrolidinium- functionalAntimicrobial effect of two highly water-soluble decaized fullerenes compound BF6 demonstrated high levels of cationic fullerenes LC14 (C60 [> M(C3 N+ dark toxicity against S. aureus, Mashino et al. showed that 6 C3 )2 ]) was and LC17 (C70 [> M(C3 N+ cationic fullerenes could inhibit the growth of E. coli and 6 C3 )2 ]) were applied for comparison in the PDT-killing of the Gram-positive S. aureus.66 S. aureus by interfering with the respiratory chain.110 111 The decacationic arms attached to these fullerenes affiliThis data suggests that photoactivated fullerenes may have somewhat different sites of action in bacteria compared to ated the rapid binding to the anionic residues of bacterial 1930
cell walls. The large number of ionic groups dramatically enhanced water solubility of these compounds. The data showed interesting differences between the photoactivity of decacationic fullerene compounds that differ only in the number of carbon atoms in the fullerene cage. For Gram-positive bacteria C60 [> M(C3 N+ 6 C3 )2 ] was better at photokilling than C70 [> M(C3 N+ 6 C3 )2 ], while for Gramnegative bacteria and for cancer cells the opposite was the case, in that C70 [> M(C3 N+ 6 C3 )2 ] was better at photokilling • than C60 [> M(C3 N+ 6 C3 )2 ]. The results of ROS (HO or + 1 O2 ) generation demonstrated that C60 [> M(C3 N6 C3 )2 ] produced more 1 O2 while C70 [> M(C3 N+ 6 C3 )2 ] produced more HO• . This finding offers an explanation of the preferential killing of Gram-positive bacteria by LC14 and the preferential killing of Gram-negative bacteria by LC17. This finding is in agreement with our previous report that Type II ROS, i.e., singlet oxygen, 1 O2 , are better at killing Gram-positive bacteria than Type I ROS, i.e., hydroxyl radicals, HO• , while the reverse is true for Gram-negative bacteria (HO• is better at killing than 1 O2 ). The hypothesis is that 1 O2 can diffuse more easily into porous cell walls of Gram-positive bacteria to reach sensitive sites, while the
less permeable Gram-negative bacterial cell wall needs the more reactive HO• to cause real damage.114 115 Antimicrobial Effect In Vivo The absorption spectrum of fullerenes is, in addition to substantial UV absorption, mainly in the blue and green visible wavelengths. This property actually limits the application of fullerene in clinical disorders, once the penetration of short wavelength light into tissue is relatively poor; however, fullerenes may still be useful as antimicrobial PS for the treatment of relatively superficial infections, where the light does not need to penetrate deeper than 1 mm. A fullerene-based PS (BF6) with tricationic charges provided by quaternized dimethylpyrrolidinium groups was found to be an effective against Gram-positive bacteria, Gram-negative bacteria and fungal yeast in vitro.75 To investigate if the high degree of in vitro activity could translate into an in vivo antibacterial PDT effect, our lab81 continued to test BF6 in two potentially lethal mouse models of wounds infected with two Gram-negative bacteria (P. aeruginosa and P. mirabilis), respectively. Compared to Gram-positive bacteria, many
Delivered by Ingenta to: University of South Carolina IP: 46.161.56.113 On: Mon, 23 May 2016 08:28:44 Copyright: American Scientific Publishers
Figure 9. BF6-PDT and tobramycin treatment of Pseudomona aeruginosa wound-infected mice. (A) Representative bioluminescence images of P. aeruginosa-infected mice (captured immediately postinfection, immediately post-treatment and 24 h posttreatment), 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 combination of 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 in Figure 4(A); no treatment control (n = 10); PDT alone (n = 12); Tobr alone (n = 2); PDT plus Tobr (n = 10). PDT: Photodynamic therapy; Tobr: Tobramycin. Reprinted with permission from [81], Z. Lu, et al., Photodynamic therapy with a cationic functionalized fullerene rescues mice from fatal wound infections. Nanomedicine (Lond.), 5, 1525 (2010). © 2010, Future Science. J. Biomed. Nanotechnol. 10, 1918–1936, 2014
needed in vivo than in vitro to achieve a certain loss of bioluminescence. The fullerene-mediated PDT succeeded in saving the life of mice whose wounds were infected with P. mirabilis and could be combined with a suboptimal dose of antibiotics to save mice with P. aeruginosa wound infections. These exciting results shown in Figure 9 indicated that fullerene-mediated PDT could either treat wounds infected with virulent species of Gram-negative bacteria or be able to synergize with a suboptimal antibiotic regimen to prevent regrowth and produce significantly higher survival.81 In the case of the 3rd-degree burns, they are particularly susceptible to bacterial infection as the barrier function of the skin is destroyed, the dead tissue is devoid of Figure 10. Representative bioluminescence images from host-defense elements, and a systemic immune suppresmice with Escherichia coli burn infections (day 0) and treated sion is a worrying consequence of serious burns. Furtherwith successive fluences of photodynamic therapy or UVA more, the lack of perfusion of the burned tissue means that light alone. (A) UVA control; (B) LC17 + UVA light; and (C) LC18 + UVA light. There was no significant reduction in systemic antibiotics are generally ineffective.116 Although bioluminescence after application of either LC17 or LC18 excision and skin grafting is now standard treatment for without light exposure as a dark control. Reprinted with the 3rd-degree burns,117 superimposed infection is still a permission from [119], L. Huang, et al., Antimicrobial photodymajor problem. Patients with Gram-negative burn infecnamic therapy with decacationic monoadducts and bisadducts tions have a higher likelihood of developing sepsis than of [70]fullerene: In vitro and in vivo studies. Nanomedicine (Lond.) (2013). © 2013, Future Science. Gram-positive infections. Topical antimicrobials are the mainstay of therapy for burn infections and PDT may have Gram-negative bacteria are much more difficult to be a major role to play in the management of this disease.118 photo-inactivated, and tend to produce systemic sepsis We found in previous study a decacationic fullerene after developing infections in wounds. Higher concentraLC17 (C70 [> M(C3 N+ 6 C3 )2 ]) was effective at mediating by Ingenta to: University of South Carolina the photokilling of Gram-negative bacteria in vitro.66 tions of PS and higher fluences Delivered of light (180 J/cm2 ) were IP: 46.161.56.113 On: Mon, 23 May 2016 08:28:44 Copyright: American Scientific Publishers
Figure 11. Representative bioluminescence images from mice with Acinetobacter baumannii burn infections and treated with photodynamic therapy, UVA light alone or absolute control, captured day 0 (before photodynamic therapy) and then daily for 6 days. (A) Absolute control; (B) UVA control + 15% DMA; (C) LC17 + 15% DMA; (D) LC18 + 15% DMA; (E) LC17 + 15% DMA + UVA light; and (F) LC18 + 15% DMA + UVA light. Reprinted with permission from [119], L. Huang, et al., Antimicrobial photodynamic therapy with decacationic monoadducts and bisadducts of [70]fullerene: In vitro and in vivo studies. Nanomedicine (Lond.) (2013). © 2013, Future Science.
We synthesized a new compound C70 [> M(C3 N6 + C3 )2 ] [> M(C3 N6 C3 )2 ] (LC18) with the same decacationic side chain plus an additional deca-tertiary amine groups against Gram-negative bacteria. A mouse model of the thirddegree burn infection with bioluminescent Gram-negative bacteria was used to test the in vivo effectiveness of the therapeutic approach using the UVA excitation.119 The data shown in Figures 10 and 11 suggested that the attachment of an additional deca(tertiary-ethylenylamino) malonate arm to C70 , producing LC18, allowed the moiety to act as a potent electron donor and increased the generation yield of hydroxyl radicals under UVA illumination. This is consistent with the reported phenomena of photoinduced intramolecular electron transfer from the tertiary amine moiety to the fullerene cage in polar solvents, including water, at the short excitation wavelength. With the availability of ten tertiary amine moieties, each capable of donating one electron to the C70 cage, LC18 may function as an electron-rich precursor for hydroxyl radical production that demonstrated a new approach in enhancing HO• radical killing of pathogenic bacteria in contrast to the more common 1 O2 -killing mechanism.
Acknowledgments: Long Y. Chiang was supported by US NIH grant R01CA137108. Michael R. Hamblin was supported by US NIH grant R01AI050875. REFERENCES
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text:
The New Rules of Angel Investing
The capital required to launch a tech startup can be 10% of what it was 10 years ago.
By Ilan MochariSenior writer, Inc.@IlanMochari
Today's capital climate is a fertile one for angel investors--and the entrepreneurs hoping to court them.
So says Ilya A. Strebulaev, an associate professor of finance at Stanford Graduate School of Business.
"Thanks in part to the plunging cost of technology, angels have enjoyed a growing competitive advantage in funding very early stage companies," he writes on the Stanford Graduate School of Business site.
Specifically, a startup that would've needed $5 million to launch 10 years ago can often launch for $500,000 today, thanks to open-source software and cloud computing, he adds. That means the time is right for entrepreneurs to court angels, who usually prefer smaller deals.
So how can you persuade angels to invest in your business? Strebulaev offers several pointers:
1. Look close to home. Even in a virtual era, it's important for investors to locate near their investments. "Most angel investments are within 50 miles of the investor, and angel networks are often in geographic clusters," writes Strebulaev.
2. Angels will hunt for the fatal flaw in your plan. "You may have a perfectly reasonable plan for a $10 million business. But angel investors are looking for companies that can ramp up to $50 million or $100 million in revenues," he writes.
The upshot? Aim high. And build barriers to entry.
"Angels want 'disruptive' ideas that will upend existing business models. If your idea is disruptive, however, can you hold on to it? Can copycats and incumbents jump in as soon as you've shown the way?"
3. Your team may be more important than your concept--or even you. "Angel investors want people who can execute the plan. They will place heavy weight on not only your track record, but also your management team," he writes. Judging your credentials is just one part of it. Angels will also try to assess if your team can stay tight-knit through adversity.
Strebulaev's article offers four more pointers on angel investors, based on his studies and firsthand observations.
Published on: Dec 21, 2013

redpajama_set_name: RedPajamaCommonCrawl