Fullerene coated surfaces and uses thereof

Substrates having a surface coated with fullerene and a substance attached thereto are disclosed. Cell culture substrates having a fullerene-coated surface are useful in methods of growing cells on the fullerene-coated surface. Methods of preparing cell culture substrates for cell attachment and growth by coating a surface with fullerene are provided. Cells can be grown on a fullerene-coated surface in the presence of a substance such as a cytokine, growth hormone or a drug, to evaluate the interaction between the substance and the cells. Methods for increasing cell membrane premability and for introducing a substance, such as a DNA or RNA vector, into a cell are also provided.

BACKGROUND OF THE INVENTION 
As a class, the recently discovered third allotropic form of carbon is 
termed fullerenes, and the prototypical form of this class is a spherical 
hollow molecule containing 60 carbon atoms, i.e., C.sub.60, that is termed 
buckminsterfullerene (Kratschmer, W., et al., Nature 347:354-357 (1990); 
Ajie, H. et al., J. Phys. Chem., 94:8630-8633 (1990); and Diederich, F., 
et al., Science. 252:548-551 (1991)). The fullerene family includes 
asymmetrical forms, such as C.sub.70, as well as cylindrical fibers 
nicknamed buckytubes. In C.sub.60, hexagons and pentagons of carbon link 
together in a coordinated fashion to form a hollow, geodesic dome with 
bonding strains equally distributed among 60 carbon atoms. Some of the 
electrons are delocalized over the entire molecule, similar to benzene. 
However, benzene is flat and many of its derivatives tend to stack in flat 
sheets. Spherical C.sub.60 adds a new dimension to the chemistry of 
aromatic compounds. 
C.sub.60 has been shown to be one of the most chemically versatile 
molecules known. Chemists have produced fullerene derivatives by adding 
carbons to the C.sub.60 sphere while maintaining (in some instances) the 
aromatic electron structure. C.sub.60 has been reported to readily accept 
free radicals and may be useful in polymerization processes. Other 
potential applications include commercial basics such as catalysis as well 
as superconductivity and ferromagnetism. C.sub.60 easily accepts 
electrons. Solid fullerene can also be doped with an alkali metal such as 
potassium, and these compounds are called fullerides. Although pure 
C.sub.60 is an insulator, some fullerides are semiconductors or 
superconductors; for example, K.sub.3 C.sub.60 is a superconductor. 
C.sub.60, a naturally hollow molecule, has been proposed as a host for 
nuclides. In addition, unmodified fullerenes are insoluble in water, 
suggesting that they may react very little with biological tissue. In the 
presence of light and oxygen, the C.sub.60 molecule can pass its 
excitation energy onto nearby oxygen molecules, creating a short-lived, 
reactive form of oxygen called singlet oxygen. 
SUMMARY OF THE INVENTION 
This invention pertains to substrates having a surface coated with 
fullerene and to the use of such substrates for immobilization and/or 
manipulation of substances on the surface. The fullerene-coated surface 
can be used to immobilize a wide variety of biological materials such as 
cells, macromolecules and drugs. In one embodiment, a surface of a 
substrate is coated with fullerene and used for attachment and growth of 
cells. Cell culture substrates having a fullerene-coated surface are 
useful in methods of growing cells on the fullerene-coated surface. 
Methods of preparing cell culture substrates for cell attachment and 
growth by coating a surface with fullerene are disclosed. Cell culture 
substrates having a fullerene-coated surface can be conditioned for cell 
attachment and growth by growing a first population of cells on the 
fullerene-coated surface and exposing the cells to a solvent, such as 
sulfolane combined with 7% perchloric acid and 3% water, designated herein 
as SPCA, to remove the cells from the fullerene-coated surface. 
Subsequently, cell material and solvent is removed from the surface to 
produce a regenerated fullerene-coated surface. A second population of 
cells can then be grown on the regenerated fullerene-coated surface and 
maintained under conditions appropriate for cell growth. Alternatively, 
the fullerene-coated surface can be conditioned for cell attachment and 
growth by coating the fullerene-coated surface with a cell attachment 
factor. 
The invention also provides methods of growing cells on fullerene-coated 
surfaces in the presence of a substance such as a cytokine, antigen, 
hormone or drug, and to evaluate the interaction between the substance and 
the cell. Methods for damaging the plasma membrane, for increasing cell 
membrane premability, and for introducing substances into a cell are also 
provided. Such methods comprise first attaching cells to a cell culture 
substrate having a fullerene-coated surface and maintaining the cells 
under conditions appropriate for cell growth. The cells are then 
illuminated with light in the presence of oxygen to damage the cell 
membrane and thereby increase cell membrane permeability. Subsequently, or 
prior to illumination, the cells are contacted with a substance to be 
introduced into the cells. The increased cell membrane permeability allows 
the substance to be introduced into the cell. The method is useful for 
transfecting a cell with, for example, a vector comprising DNA or RNA.

DETAILED DESCRIPTION OF THE INVENTION 
Fullerenes are commonly derived by contact-arc vaporization of a graphite 
rod, which results in the formation of raw soot. The raw soot produced by 
this process primarily comprises a mixture of two fullerenes, C.sub.60 and 
C.sub.70 in a ratio of about 10 to 1 respectively, accounting for about 5 
to 10% of the total soot. Other methods of deriving fullerene-containing 
soot, primarily from sooting flames, also exist. Fullerenes such as 
C.sub.60, C.sub.70, C.sub.76, C.sub.78, C.sub.84, etc., can be deposited 
on a substrate alone following purification, or as a mixture with one or 
more fullerenes. 
Fullerenes can be recovered from raw soot by extraction with organic 
solvents, such as benzene or toluene, followed by precipitative or 
evaporative deposition of the fullerenes on a surface of a substrate and 
solvent removal. Alternatively, fullerenes can be recovered by sublimation 
under vacuum with fullerene vapor being condensed as a film upon a 
relatively cool substrate surface. Fullerenes can also be coated on the 
surface of a substrate by ion-sputtering of purified fullerene or raw 
unprocessed fullerene-containing soot. 
Fullerenes coated to a surface of a substrate are useful for immobilizing 
and/or manipulating substances on the surface. The fullerene-coated 
surface can be used to immobilize biological materials including cells, 
macromolecules, drugs, aromatic molecules, and aliphatic substances. 
In one embodiment, fullerene coated surfaces are used as a substrate for 
cell growth. Cells are cultured in an appropriate media on a 
fullerene-coated surface of a cell culture substrate to facilitate cell 
attachment and growth. A surface of a cell culture substrate such as a 
ceramic, a polymer or a carbon matrix can be coated with fullerenes and 
used in methods of cell culture. Preferred ceramic cell culture substrates 
comprise glass or quartz. Polymer substrates useful for cell culture 
include polystyrene, polypropylene, polyhydroxyethyl methylacrylate, 
polyethylene terephthalate, polytetrafluoroethylene and nylon. A preferred 
polymer for forming a cell culture substrate is polystyrene. Useful cell 
culture substrates include devices such as cell culture flasks, cell 
culture dishes, cell culture microcarriers, cell culture macrocarriers, 
cell culture films and cell culture fibers. 
Cell culture substrates having a fullerene-coated surface are useful in 
methods of growing cells. Cells are cultured on a cell culture substrate 
in an appropriate media (e.g., Dulbecco's Modified Eagle Media, CMRL 
Media, Minimum Essential Media or RPMI Media 1640 for mammalian cell 
lines) which may contain factors necessary for cell proliferation and 
viability, including animal serum (e.g., fetal bovine serum), hormones, 
protein growth factors, and antibiotics (e.g., penicillin, streptomycin). 
Additional factors, such as extracellular matrix components (e.g., 
collagen, fibronectin, and polylysine) or attachment peptides (e.g., RGD; 
Arg-Gly-Asp) can be coated on the cell culture substrate to enhance cell 
adhesion and growth. The cells are maintained under conditions necessary 
to support cell growth, for example an appropriate temperature (e.g., 
37.degree. C.) and atmosphere (e.g., air plus 5% CO.sub.2). 
Illumination of a fullerene in solution with absorbing light in the 
presence of molecular oxygen generates highly reactive singlet oxygen 
(.sup.1 O.sub.2) by a semi-catalytic process without concurrent damage to 
the fullerene (Arbogast, J. W., et al., J. Phys. Chem. 95:11-12 (1991)). 
Substances, such as cells or macromolecules can therefore be attached to a 
fullerene-coated surface and illuminated with light in the presence of 
oxygen to induce .sup.1 O.sub.2 damage. When cells are attached to a 
fullerene-coated surface, the reactivity of .sup.1 O.sub.2 is such that it 
is unlikely to diffuse beyond the attached cell membrane (Moan, J., J. 
Photochem. Photobiol. B: Biology 6:343-344 (1990); Suwa, K., et al., 
Biochem Biophys. Res. Comm. 75:785-792 (1977)). Thus, the predominate 
oxidative reactions are likely to be peroxidations and cycloadditions of 
.sup.1 O.sub.2 at carbon-to-carbon double bonds, resulting in increased 
cell membrane permeability. The length of illumination, intensity and 
wavelength of the light can be selected to quantitatively control the 
membrane induced damage. This technique is useful to study cell membrane 
composition e.g., cholesterol content, low- vs high-density lipoprotein 
interactions and the effect of oxidative damage on the cell membrane. 
In addition, selective increases in cell membrane permeability allow the 
introduction of substances (e.g., a vector) into the cell. Cells attached 
to a fullerene-coated surface can be cultured with a substance to be 
introduced into the cell prior to, simultaneously with, or following 
illumination with light in the presence of oxygen. For example, membrane 
porosity can be manipulated to accommodate the entry of vectors, 
transfecting DNA, antibodies and other proteins, DNA pool intermediates, a 
drug, etc. As described in detail in the examples, Chinese Hamster Ovary 
(CHO) cells illuminated with light in the presence of oxygen were observed 
to take up trypan blue, an event indicating an increased permeability of 
the cell membrane (Lichtenstein, A., J. Clin. Invest 88:93-100 (1991); 
Baker, S. S., et al., Gastroenterology 101:716-720 (1991)). In addition, 
the cells illuminated in the presence of oxygen were shown to resist 
detachment by trypsinization or contraction by air drying, indicating 
treatment-induced bonding of cell constituents to the fullerene surface. 
Singlet oxygen-induced damage to cell membranes can also be used to study 
the interaction and effects on cell growth and viability of various 
substances such as cytokines (e.g., interleukin-2), lectins (e.g., 
phytohemoglutinin), hormones, growth factors, oncogene products, 
monoclonal antibodies, ion-channel complexes, antigen receptors, DNA, RNA, 
polyethylene glycol, glycogen, drugs, aromatic molecules, steroids, 
phospholipids, long chain aliphatic substances and cell attachment 
factors. Membrane-related drug interactions can be probed, e.g., 
interactions with the antitumor drug adriamycin or with a variety of 
anesthetic agents. These substances can be co-cultured with cells on a 
fullerene-coated surface to evaluate the interaction between the substance 
and the cell. In addition, the cells can be cultured with the substance 
and illuminated in the presence of oxygen to determine the effect on the 
cell upon introduction of the substance into the cell or upon alteration 
of the cell membrane during illumination. 
The fullerene structure is overall electrophilic, thus accounting for its 
tendency to react with electron rich reagents and free radicals (Haufler, 
R. E., et al., J. Phys. Chem., 94:8634-8636 (1990); Fagan, P. J., et al., 
Science 252:1160-1161; Krusic, P. J., et al., Science 254:1183-1185 
(1991)). This free radical reactivity may allow for controlled 
derivitizations of the fullerene surface for the purpose of directing 
specific cell membrane interactions. Derivatized fullerene surfaces would 
be useful for directing specific cell attachment and growth, as well as 
manipulating membrane damage by singlet oxygen. 
An important feature of fullerenes is their ability to act as a form of 
"activated carbon". The fullerene electronic structure is a system of 
overlapping pi-orbitals, such that a multitude of bonding electrons are 
cooperatively presented around the surface of the molecule (Chemical and 
Engineering News. Apr. 8, 1991, page 59). This extensive system of 
overlapping pi-orbitals produces resonance hybrids with similarity to 
graphite, such that the longer the fullerene structure, the greater this 
similarity (Haddon, R. C., Acc. Chem. Res., 25:127-133 (1992)). As a form 
of activated carbon, fullerenes exhibit substantial van der Waals forces 
for weak interactions. These weak interactions account for the well known 
adsorptive capacity of activated carbon (Hassler, J. W., Active Carbon: 
The Modern purifier, Githens-Sohl Corp, New York, 1941; Snyder, L. R., 
Principles of Adsorption Chromatography, Marcel Dekker, lnc., New york, 
1968). The adsorptive nature of the fullerene surface may lend itself to 
additional modifications for the purpose of directing specific cell 
membrane interactions. For example, specific molecules that possess 
chemical properties that selectively bind to membranes of particular cell 
types or to particular components of cell membranes generally, e.g., 
lectins or antibodies can be adsorbed to the fullerene surface. The 
fullerene surface can be chemically modified to present specifically 
reactive groups to the cell membrane, e.g., oxidants or reductants. 
Attachment of different molecules to a fullerene surface could be 
manipulated to result in a surface which favors attachment of specific 
molecular or cell types, such as epithelial cells, fibroblasts, primary 
explants, or T-cell subpopulations by use of, for example, T-cell antigen 
receptors or antibody bound to the fullerene-coated surface. 
The adsorptive nature of fullerenes can be exploited in methods of 
enhancing cell attachment to a fullerene-coated surface. In one 
embodiment, a fullerene-coated surface is conditioned for cell attachment 
and growth by growing a first population of cells on the fullerene-coated 
surface and exposing the cells to a solvent, e.g., SPCA, to remove the 
cells from the fullerene-coated surface. Subsequently, cell material and 
solvent is removed from the surface to produce a regenerated 
fullerene-coated surface. A second population of cells is grown on the 
regenerated fullerene-coated surface by maintaining the cells under 
conditions appropriate for cell growth. Suitable solvents include 
acidified sulfolane (e.g., acidified with perchloric acid (SPCA) or with 
trifluoromethane sulfonic acid), sulfolane alone, or other saturated or 
unsaturated ring system solvents having polar hydrophilic groups, as well 
as digestive enzyme solutions. For example, it was apparent by microscopic 
observation that CHO cells grown on surfaces coated with fullerene did not 
grow as rapidly or were as tightly attached (qualitatively judged by 
degree of rounding of cells attached to the surface) as cells attached to 
fullerene coated surfaces that were regenerated with SPCA for reuse. The 
superior cell attachment observed for these regenerated fullerene surfaces 
on either glass or plastic is likely due to absorption of cell-contained 
hydrophilic and/or receptor-like moieties to the carbon surface. Moreover, 
the solfolane of SPCA may have an affinity for activated carbon which 
explains the slightly enhanced cell attachment and growth observed for 
cells on SPCA-only treated fullerene surfaces given the hydrophilic 
non-bonding sulfoxide portion of the sulfolane molecule. SPCA is highly 
acidic, and sulfolane alone, or other saturated or unsaturated ring system 
solvents having polar hydrophilic groups, may be effective in conditioning 
the fullerene surface for subsequent cell attachment and growth. Thus, 
digestion or dissolution of a specific cell type or conditioning by 
specific adsorbing molecules on a fullerene surface may result in a 
modified fullerene surface that is selective for attachment and growth of 
a specific cell type from a mixed population of cells or that results in 
an improved surface for cell attachment and growth. 
In another embodiment, a fullerene-coated surface is conditioned for cell 
attachment and growth by coating the fullerene-coated surface with an 
attachment factor prior to growing cells on the fullerene-coated surface. 
For example, CHO cells were found to have improved cell attachment on a 
fullerene-coated surface which had been treated with poly-L-lysine. The 
enhanced cell attachment is likely due to adsorption of this polymer to 
the active carbon surface. Other attachment factors, such as fibronectin 
and collagen can also be used. 
The following non-limiting examples demonstrate fullerene retrieval from 
raw soot by vapor deposition of a fullerene film on either glass or 
plastic cell culture substrates, or by evaporative deposition on glass of 
C.sub.60 dissolved in benzene. CHO cells were found to attach and grow on 
these fullerene-coated surfaces. In addition, illumination of the 
fullerene surface with light in the presence of molecular oxygen induced 
damage to the plasma membrane of the cells as indicated by the uptake of 
trypan blue and by visual observations of increased cell sticking to the 
supporting surface and by decreased symmetry of cell morphology. 
EXAMPLE 1: SURFACE SUBSTRATES AND FULLERENE SURFACE PREATION 
Purified C.sub.60 was obtained as a gift from Dr. Christopher S. Foote, 
University of California at Los Angeles. Drops of magenta colored 1 mM 
solution of C.sub.60 in benzene were spread on a 22.times.22 mm glass slip 
and evaporated leaving a yellowish gold microcrystalline residue that only 
partially covered the surface of the slip. Drops of a 1 mM solution of 
C.sub.60 in benzene were also spread on the polystyrene surface of a petri 
dish and allowed to air dry, leaving a uniform magenta color at the 
plastic surface. Two-tenths of a ml of a 500 .mu.m C.sub.60 benzene 
solution covered a glass slip edge-to-edge, and was allowed to evaporate 
at ambient conditions leaving a yellowish film of C.sub.60 interspersed 
with thicker plates of yellowish gold C.sub.60 crystals. 
Raw unprocessed fullerene-containing soot from carbon-arc vaporization of 
graphite rods was purchased from Materials and Electrochemical Research 
Corp. (Tucson, Ariz.). Approximately 0.1 to 0.2 g of this soot was 
moistened with methanol and loaded into a 0.375.times.1.00.times.0.375 
inch molybdenum boat with a lid containing a small hole. The boat was 
suspended between two copper bars in a diffusion-pumped chamber. The 
substrates of glass or plastic to be coated were suspended above the boat 
by adhesive or by a through-drilled plate that exposed the majority of the 
substrate surface. Pressure was reduced to ca. 5.times.10.sup.-5 Torr, at 
which point an AC current was driven through the boat, electrothermally 
heating it above the sublimation temperature of the fullerene contained in 
the raw soot. The rate and thickness of vapor deposition on the substrate 
were monitored using an oscillating quartz crystal. Conductive heating of 
raw soot in a vacuum chamber placed in a 550.degree. C. sand bath also led 
to sublimation and deposition of fullerene surface on glass slide 
segments, glass slips, and polystyrene substrates suspended above the 
soot. 
Glass slips used as substrate were no. 2 thickness from Corning; glass 
slides were 1 mm thick from Fisher. In one case, 2 slips and one slide 
were not thoroughly cleaned, i.e., had a barely discernible residue that 
appears to be resident on new slides and cover slips, prior to exposure to 
fullerene vapor; after fullerene exposure the slide was sectioned to 
pieces approximately the size of the slips. The latter two slips and slide 
had ca. 3 mm margins on two sides where fullerene deposition was not 
allowed, so as to afford the ability to distinguish differences in cell 
response on the same support surface. In a second case, 17 glass slide 
segments the size of slips were extensively cleaned by a chloroform rinse 
and polish, followed by ultrasonic cleaning in methanol prior to fullerene 
exposure. Several of these segments had masked margins to preclude 
fullerene deposition. In a third case, plastic dishes were taken as 
received from the manufacturer and subjected to fullerene vapor 
deposition; one dish type was unmodified polystyrene (Falcon 
bacteriological petri dish, no. 4-1008-3) and a second dish type was 
polystyrene made hydrophilic by proprietary superficial treatment with 
negatively charged groups, e.g., carboxyls and carbonates (information 
from Technical Service, Becton Dickinson Labware, Falcon Division) (Falcon 
tissue culture dish, no 4- 3001-3). One of the polystyrene dishes had a 4 
mm mask extending across its center where fullerene deposition was 
prevented. 
One petri dish and one tissue culture polystyrene dish prepared with 
fullerene surfaces were treated with poly-L-lysine hydrobromide (PL) 
(Sigma P-2636, Type VII-B; approximate MW 60,000). A 100x stock solution 
was prepared by dissolving 34 mg of PL in 3.4 ml borate buffer (5.7 g 
sodium borate in 100 ml distilled water, adjusted to pH 8.4 with HCl), 
filter sterilized and aliquots frozen until use. Dilution of 1:100 was 
then made in sterile borate buffer. The surface of the fullerene-prepared 
dishes were covered with 1.times.PL solution, held 2 hours at 33.degree. 
C., and rinsed exhaustively with PBS prior to seeding cells as described 
below. 
The fullerene surface of one glass slip was directly scanned in a 
Perkin-Elmer Lambda 3A UV/VIS spectrophotometer, and then dissolved in 
benzene and this solution again scanned in the spectrophotometer. The 
integrity of the fullerene surface was qualitatively ascertained by visual 
and microscopic examination. Durability of the surface was ascertained as 
the continued integrity following autoclaving (glass substrate), exposure 
to and agitation with aqueous solutions as well as a solution of sulfolane 
containing 10% reagent grade perchloric acid (SPCA), and repetitive 
culturing of cells on the same fullerene surface following regeneration by 
exposure to SPCA. Vapor deposition of fullerene onto polystyrene or 
uncleaned glass resulted in a tightly attached film. Vapor deposition onto 
the freshly ultrasonically cleaned glass slide segments, however, provided 
a film that initially cracked upon subsequent autoclaving and was washed 
off from some segments by a water rinse; however, after a 3 month storage 
period under dark ambient conditions the remaining autoclaved segments 
were found to have their fullerene film tightly bonded to the glass 
substrate and thus resisted removal by vigorous rinsing or incubation with 
cells. It is thought that slow complexation with glass, or with charge 
groups in contaminants associated with the glass substrate, affords this 
bonding of fullerene films onto the ultrasonically cleaned glass surface. 
EXAMPLE 2: CELL CULTURE AND ILLUMINATION OF CELLS ON FULLERENE COATED 
SURFACES 
A Chinese Hamster Ovary (CHO) cell line designated AA8 was provided by Dr. 
Larry Thompson (Lawrence Livermore National Laboratory) (Hoy, C. A., et 
al., Mutation Res. 130:321-332 (1984)). About 3.times.10.sup.5 cells were 
seeded into the prepared 35 mm dishes or dishes containing the prepared 
slips or slide segments and grown in alpha-MEM containing 10% fetal bovine 
serum, penicillin and streptomycin antibiotics and 40 nmol/liter 
17-beta-estradiol (compete media) using an incubator at 37.degree. C. 
equilibrated with air plus 5% CO.sub.2. Slips, slide segments, and plastic 
dishes with fullerene surfaces regenereated for reuse by removing cells 
with treatment of SPCA contact for 2 hours at 33.degree. C., followed by 
extensive washing with phosphate buffered saline, pH 7.0 (PBS), drying and 
autoclaving (glass only), and reseeding with CHO cells in complete media 
for regrowth. 
Slips were placed in 32 mm glass dishes over a small stir bar, and were 
overlayed with complete media. This dish was placed on the bottom of a 
can-type vacuum chamber with a quartz window on top, and the media was 
stirred. For variable oxic control, two cycles of pumping the chambers to 
52 mm Hg followed by back-filling with either 19% or 95% 0.sub.2 (plus 5% 
C0.sub.2 balance N.sub.2) were completed at room temperature in 1 minute, 
after which chambers were closed and samples held for an additional 10 
minutes. For hypoxic equilibration, the chamber with cell sample was 
subjected to five cycles of pumping and back-filling with N.sub.2 plus 5% 
CO.sub.2 over the course of 20 minutes, followed by holding for an 
additional 10 minutes. After atmospheric manipulation, chambers were 
two-thirds immersed in a temperature-controlled water bath and held 7 
minutes for equilibration at 35.degree. C. Cells were then illuminated for 
10 or 30 minutes through the window of the chamber using a Kodak 
Ektagraphic IIIB slide projector operating a 300 Watt incandescent lamp 
with output as previously described (Richmond, R. C., J. O'Hara, 
Photochem. Photobiol., in press (1992)) such that irradiance to the cells 
was 98 mW/cm.sup.2. Temperature rise during illumination plateaued after 
10 minutes at 35.degree. C., as separately monitored by a thermocouple 
placed in the sample media. 
Following illumination, cells grown to confluency on initially deposited 
fullerene on glass slips were trypsinized from the fullerene surface using 
standard 1.times.porcine trypsin (ca. 1500 BAEE units/ml PBS, 30 minutes 
at 33.degree. C.) and triturated to single cells. These cells were counted 
for trypan blue exclusion in a PBS solution of 0.08% trypan blue. One 
hundred cells (based on the trypan blue exclusion count) were plated in 
complete media and grown for 7 days at 37.degree. C., after which time 
colonies were stained with methylene blue and counted. Cells resisting 
removal from the fullerene surface by 1.times.trypsin were found to take 
up trypan blue, and also to be removed by incubation with an additional 
exposure to 50.times.trypsin plus 0.02% disodium 
ethylenediaminetetraacetate (EDTA) for about 60 minutes at 33.degree. C. 
Alternatively, cells grown on an SPCA-regenerated fullerene surface on 
glass slips or slide segments were exposed directly after illumination to 
0.08% trypan blue in PBS for 20 minutes at 33.degree. C. followed in order 
by: 5 rinses in PBS; a 2 hour exposure to phosphate buffered 3.3% 
formaldehyde; a rinse in water; air drying; and microscopic assessment. In 
a third case, cells were similarly exposed to trypan blue and PBS rinsing 
after illumination, but were then treated with SPCA for 2 hours at 
33.degree. C., after which the fullerene surface was triturated and the 
SPCA solution of photometrically analyzed for trypan blue concentration at 
568 nm using an extinction coefficient of 7.7.times.10.sup.4 liter/mol.cm 
determined from trypan blue dissolved in SPCA alone. 
EXAMPLE 3: ASSESSMENT OF CELL MEMBRANE PERMEABILITY FOLLOWING ILLUMINATION 
OF CELLS ON SURFACES PREED WITH PURIFIED FULLERENE 
CHO cells were grown over the dispersed residue of yellowish gold 
microcrystalline C.sub.60 left on the glass slip by evaporating drops of a 
1 mM C.sub.60 benzene solution, and were then illuminated under 95% 
O.sub.2 plus 5% CO.sub.2 for 30 minutes (147 J/cm.sup.2). A small amount 
of foaming and cell clumping was noted when cells were then trypsinized, 
suggesting some membrane damage during illumination. Similar observations 
of minor membrane damage were made for CHO cells grown on a polystyrene 
surface exposed to a 1 mM solution of C.sub.60 in benzene, treated with 
poly-L-lysine, and illuminated as described above. Although the surface of 
this plastic appeared dry after exposure to the C.sub.60 solution, the 
magenta color of the solution was preserved, suggesting that a C.sub.60 
solution was maintained within the polymeric skein of the plastic. The 
continuous film of C.sub.60 interspersed with crystalline plates resulting 
from evaporating 0.20 ml of 500 .mu.M C.sub.60 in benzene also supported 
confluent CHO cell growth, and 5 minutes of illumination in air led to 
cell damage as determined by increased cell sticking to the supporting 
surface and by decreased symmetry of cell morphology assumed to reflect 
damage to plasma membrane. 
EXAMPLE 4: ASSESSMENT OF CELL MEMBRANE PERMEABILITY FOLLOWING ILLUMINATION 
OF CELLS ON A GLASS SUBSTRATE PREED BY FULLERNE VAPOR DEPOSITION 
Two uncleaned glass slips (Slip #2 and Slip #3) were prepared with an 
approximate 500 nm thickness of fullerene by electrothermal vapor 
deposition from raw unprocessed carbon-arc soot, such that 3 mm glass-only 
margins were maintained on two edges of the slide. These slips were then 
autoclaved. The golden bronze fullerene surface was microscopically 
determined to be smooth and unflawed both before and after autoclaving. 
About 10.sup.6 CHO-AA8 cells were seeded and grown to confluency, and 
this was noted to take 3 days on these fullerene-coated slips rather than 
the two days on the unprocessed glass slip (Slip #1). 
Cells on Slip #1 were not further treated, whereas cells on Slips #2 and #3 
were brought to 95% O.sub.2 plus 5% CO.sub.2 in complete media and 
illuminated at 37.degree. C. for 10 minutes (59 J/cm.sup.2) and 30 minutes 
(177 J/cm.sup.2), respectively. Cells were then trypsinized from all slips 
in standard fashion. Slip #1 afforded an excellent single cell suspension 
with minimal trituration, yielding a total of 5.1.times.10.sup.6 cells. 
Slips #2 and #3 afforded a clot of cells that lifted off the slip as a 
sticky mass that then provided only a partial yield of single cells (61% 
and 41% of the control number, respectively) in suspension after extensive 
trituration. 
Trypan blue exclusion was determined, and 100 cells were plated in 
triplicate based on the trypan blue exclusion counts, and colonies were 
then scored. Results are shown in Table I. 
TABLE I 
______________________________________ 
% Trypan Blue 
% Plating Surviving 
Slip # 
Uptake Efficiency (SD) 
Fraction (SD) 
______________________________________ 
1 3 87 (10) 1.00 (0.15) 
2 7 56 (7) 0.64 (0.08) 
3 8 54 (6) 0.62 (0.07) 
______________________________________ 
It is clear that the fraction of single cells recovered from the sticky 
mass of illuminated trypsinized cells were only slightly adversely 
affected in regards to membrane permeability (judged as trypan blue 
uptake) and inability to form colonies relative to control cells. Also 
clear is the fact that membrane damage did result from illumination of 
cells on fullerene surfaces due to the stickiness observed after 
trypsinization and the incomplete recovery of cells following extensive 
trituration. 
Slips #2 and #3 were observed to retain patches of cells on the fullerene 
surfaces after 1.times.trypsin treatment. These 1.times.trypsin resistant 
cells were dissociated from Slip #2 by an additional 60 minutes incubation 
at 37.degree. C. with 50.times.trypsin plus 0.02% EDTA. These resistant 
cells on Slip #3 were also found to take up trypan blue when covered with 
a 0.08% solution, as shown in FIG. 1 (magnification 480X) where the 
fullerene surface was scratched next to the photographed patch of cells in 
order to show the appearance and integrity of these vapor deposited 
fullerene surfaces. 
Both Slips #2 and #3 were regenerated by exposure to SPCA, followed by 
rinsing and air drying. Slip #2 was scanned directly in the 
spectrophotometer (FIG. 2; Ca 50nm thickness vapor deposited fullerene), 
and the fullerene was then dissolved in 1 ml of benzene and the spectrum 
of this solution was also determined (FIG. 3) and compared to that of 
authentic C.sub.60 (FIG. 4; a 25 .mu.mol/liter solution of C.sub.60 in 
benzene). The thickness of the fullerene surface was too great to resolve 
spectral structure, but for a peak at 337 nm, corresponding to a peak 
reported for C.sub.60 deposited on quartz (Kratschmer, W., et al., Nature 
347:354-357 (1990)). The benzene solution was judged to contain a complex 
mixture of fullerenes, primarily of C.sub.60, with the peak at 277 nm 
being specific for C.sub.60. From the latter absorbance value, and using 
an extinction coefficient of 6.04.times.10.sup.4 liter/mol.cm calculated 
from FIG. 4, the amount of C.sub.60 contained on Slip #2 was estimated at 
about 8 .mu.g. 
Slip #3 was regrown with CHO cells, and these were subjectively noted to 
grow more rapidly and with better spreading (i.e., attachment), on the 
surface than when the fullerene surface was directly used. When confluent 
with cells, the slip was equilibrated under 19% O.sub.2 plus 5% CO.sub.2 
(balance N.sub.2), illuminated for 30 minutes (177 J/cm.sup.2), and then 
immediately exposed to trypan blue. Thereafter, cells were rinsed, fixed 
with buffered formaldehyde, rinsed again, and air dried. FIG. 5 shows the 
effect of illumination on the subsequent trypan blue uptake by these 
cells. Cells on the left half of FIG. 5 grew on the non-coated surface of 
the slip margin; cells on the right half grew on the deposited fullerene 
surface. Illumination in the presence of O.sub.2 led to extensive membrane 
damage for cells only on the prepared surface such that a) substantial 
trypan blue uptake was observed (i.e., extensive membrane permeability), 
and b) cells on the prepared surface were "fried" in place (i.e., probably 
glued down by a reacted residuum) and did not pull up into refractile 
forms otherwise achieved during the drying process after fixation. 
The refractile cells on the non-prepared glass surface in FIG. 5 were not 
damaged as judged by lack of trypan blue uptake and by morphologic 
appearance that matched that of cells grown on a fullerene surface 
deposited on a glass slide segment without illumination (FIG. 6). 
Furthermore, the absolute dependence of O.sub.2 on the light-induced 
damage to cells on the fullerene surface in FIG. 5 was established by 
regenerating Slip #3 with SPCA treatment, regrowing cells, equilibrating 
under 95% N.sub.2 plus 5% CO.sub.2, and illuminating for 30 minutes (177 
J/cm.sup.2) (FIG. 7). In this situation, trypan blue uptake was not 
observed and cells appeared as in FIG. 6 after fixation and air drying. 
Trypan blue uptake by cells was quantitated with and without illumination. 
Slip #3 was regenerated again with SPCA treatment and cells were grown to 
confluency as usual. Illumination conditions that led to FIG. 5 were 
duplicated (19% O.sub.2, 177 J/cm.sup.2), followed then by the same 
exposure to trypan blue. After rinsing, cells were dissolved by SPCA, and 
the cell uptake of trypan blue was quantitated by spectrophotometry 
(spectrum shown in FIG. 8) to equal 1.1 Pg/cell using the extinction 
coefficient of 7.70.times.10.sup.4 liter/mol.cm calculated at 582 nm for 
trypan blue dissolved in SPCA alone (spectrum shown in FIG. 9). Comparing 
FIGS. 8 and 9 it is noted that the major peak for trypan blue in the cell 
solution is blue-shifted by 14 nm. Slip #3 was again regenerated and cells 
again grown to confluency and identically treated except that light 
exposure was reduced by a third to 59 J/cm.sup.2 ; trypan blue uptake was 
found to be 0.26 pg/cell, or about a four fold reduction, indicating an 
illumination dose response of cell membrane damage leading to trypan blue 
uptake. Recovery of trypan blue from nonilluminated confluent monolayers 
of cells growing on glass directly or on fullerene deposited on a slide 
segment was the equivalent of 0.08 pg/cell in both cases; this low level 
of trypan blue is probably recovered from the interstitial space of the 
cell monolayer. 
Results above used fullerene surfaces on glass slips or slide segments that 
had not been scrupulously cleaned prior to fullerene vapor deposition. The 
resulting fullerene surface was uniform and durable with respect to fluid 
triturtations and autoclaving. An additional set of 17 glass slide 
segments was scrupulously cleaned by first rinsing in chloroform, 
polishing, and finally subjecting to ultrasonic cleaning in a methanol 
bath directly prior to fullerene vapor deposition. The integrity of the 
fullerene film was initially as good as that obtained on non-specially 
cleaned glass substrate. However, autoclaving produced multiple fractures 
of the surface that was also then susceptible to loss by gentle rinsing. 
In addition, gentle contact with fluids served to fracture and wash away 
substantial sections of the fullerene surface on non-autoclaved segments. 
Autoclaved slide segments were found to have reestablished a bonded 
fullerene surface. However, after three months of storage in the dark 
under ambient conditions, either slow complexation of fullerene to the 
cleaned glass substrate, or reemergence of containments or charged groups 
on the glass substrate originally removed by cleaning and important to 
fullerene complexation, or both, are hypothesized to explain the latter 
observation. 
EXAMPLE 5: ASSESSMENT OF CELL MEMBRANE PERMEABILITY FOLLOWING ILLUMINATION 
OF CELLS ON A POLYSTRYENE SUBSTRATE PREED BY FULLERENE VAPOR DEPOSITION 
Polystyrene dishes, termed petri dishes, are hydrophobic such that water 
droplets bead up on their surface. Petri dishes that are treated with 
negatively charged chemical groups in a commercial proprietary process are 
termed tissue ware dishes and are hydrophilic such that water droplets 
spread out in a film on their surface. Mammalian cells will attach to the 
surface of tissue ware dishes, but are generally said not to attach to the 
surface of petri dishes. Fullerene was deposited on both petri dishes and 
tissue ware dishes. Following this deposition, both dishes were observed 
to be equally hydrophobic as determined by water droplet beading that was 
equivalent to that seen with petri dishes directly. Interestingly, beading 
of water droplets on vapor deposited fullerene surfaces on glass slide 
segments was less pronounced than on directly deposited fullerene on 
polystyrene, although more pronounced than on directly deposited fullerene 
on glass. It is hypothesized that long range ionic forces in the glass 
substrate are preserved to some degree across the thin fullerene film. The 
fullerene surface on both petri and tissueware dishes was found to be very 
uniform and of good durability when rinsed with liquids. 
CHO cells were seeded into both petri dishes and tissue ware dishes 
directly after fullerene deposition, and attachment and growth were 
observed under the microscope. Attachment was relatively poor. Over the 
course of 4 days at 37.degree. C. it was clear that cells were actively 
dividing, but this growth was out into suspension, not on the fullerene 
surface where the number of cells attached in both rounded and flattened 
forms equalled about a third of what would be seen on glass or tissue ware 
surfaces, and the flattened, i.e., well attached cells, approximated only 
about 10-20% of the total. Pretreating the fullerene surface with SPCA for 
2 hours at 33.degree. C. prior to growing cells resulted in better cell 
attachment. Growing cells on the fullerene surface and then regenerating 
the dish for reuse by dissolving these cells with SPCA for 2 hours at 
33.degree. C. resulted in a more hydrophilic surface and improved cell 
attachment. Dishes identically regenerated with SPCA followed by treatment 
with poly-L-lysine (as is typically done with polystyrene in order to 
create a surface favorable for cell attachment) resulted in a more 
hydrophilic surface and improved cell attachment. 
Although glass slips and slides are supplied as "precleaned" from the 
manufacturer, there is obviously some thin, unidentified deposit on the 
glass surface. Also, it is common practice not to clean "precleaned" 
microscope slides for mounting tissue sections for histological work-up 
because paraffin-blocked tissue sections do not stick as well to truly 
clean slides. It was similarly found in this work that fullerene vapor 
deposited films initially did not stick well to a scrupulously clean glass 
substrate, but that good bonding of these films did develop over three 
months of dark storage under ambient conditions. Fullerene films did stick 
rather well initially to non-cleaned clips and slide segments. 
Furthermore, fullerene films were found to stick relatively well to 
scrupulously clean polystyrene substrate. From these three latter 
observations it appears that fullerene vapor deposition requires contact 
with organic or other bonding molecules at the surface of a plastic or 
glass substrate to form bonds sufficient to withstand mechanical stress, 
such as encountered with autoclaving or triturating which are typical in 
tissue culture techniques. 
Cells seeded on glass surfaces coated with fullerene grow and attach on the 
surface better (the latter was qualitatively judged by degree of rounding 
observed for cells attached to the surface) than cells seeded on 
polystyrene coated with fullerene. Regardless of whether fullerene was 
deposited on a modified polystyrene surface (i.e., hydrophilic surface), 
the fullerene coated surface was found to be hydrophobic as indicated by 
the beading of water in these dishes. In contrast, glass slips or slide 
segments coated with fullerene were notably less hydrophobic, though not 
as hydrophilic as the glass substrate itself. It is commonly acknowledged 
that a hydrophilic surface is necessary to achieve significant mammalian 
cell attachment. Thus, it appears that a glass substrate may establish 
hydrophilic centers across a thin (ca. 50 nm estimated maximum) fullerene 
surface, and that such hydrophilicity may be established by long range 
ionic perturbations caused by monovalent and divalent cations found in 
glass but not polystyrene. Thus, the readily manipulated hydrophobic and 
hydrophilic nature of the fullerene surface may allow selection of cell 
type in culture techniques, e.g., preferential attachment of 
polymorphonuclear leukocytes which are known to selectively adhere to 
plastic such as polyethylene or polypropylene. 
Those skilled in the art will recognize, or be able to ascertain using no 
more than routine experimentation, numerous equivalents to the specific 
procedures described herein. Such equivalents are considered to be within 
the scope of the this invention and are covered by the following claims.