1. A Simple Two-Step Method for Molecular Immobilization Onto Buckminsterfullerenes
Sairaj Sajjath
1/1/2015

2. 2
Abstract
An ideal targeted drug delivery system consists of a targeting molecule that actively
targets diseased cells and a drug carrier that optimizes drug delivery/release. While both
targeting molecule and drug carrier technology have advanced greatly in recent years, no method
has been established to effectively immobilize a wide range of targeting molecules onto drug
carriers and assemble the two into a functional system. This study bridges this gap by developing
a simple, two-step method for the immobilization of a wide range of targeting molecules onto
buckminsterfullerenes, a highly promising class of nanoparticle drug carriers. This method is
centered around the bifunctional molecule 1-pyrenebutanoic acid, succinimidyl ester (PASE),
which indirectly induces immobilization through amide bond formation with amine-modified
targeting molecules and π-stacking with fullerene surfaces. In this work, aptamer AS1411 was
immobilized onto buckminsterfullerene C60; reactions were proven successful via ATR-FTIR
spectrometry. Overall, this study provides a general method to bridge advances in targeting
molecule and drug carrier technology, and in the future will allow for assembly of a wide array
of targeted drug delivery systems through a simple, standard process.

3. 3
Introduction
Buckminsterfullerenes are hollow, spherical carbon allotropes that exhibit potent
structural, mechanical, and photochemical properties [1,2]. These properties open doors to
valuable applications of the molecules, most notably in targeted drug delivery [3-15].
Targeted drug delivery systems are revolutionary medical tools that direct
pharmaceuticals specifically to diseased tissues [16,17]. These systems, which use “targeting
molecules” such as antibodies, peptides, aptamers (oligonucleotides) and small molecules to
target drug carriers to specific areas in the body, prevent healthy areas from being harmed and
allow for greater drug dosages than conventional treatments. Despite their benefits, however,
targeted drug delivery systems remain difficult to produce and are consequently unaffordable to
many patients. This is due chiefly to the difficulties in producing targeting molecules, which are
primarily produced through inefficient biological extraction and “humanization” (making
compatible with human systems) processes, and conjugating targeting molecules to drug carriers
[18,19].
The problem of complex targeting molecule production is being rapidly remedied
through synthetic targeting molecules, namely aptamers and small molecules, which offer highly
efficient, low-cost alternatives to their biological counterparts [18-21]. However, advances in this
field mean little without a reliable method to conjugate these synthetic molecules to drug
carriers. The small (1 nm diameter) size, large “loading capacity” (drugs per carrier)[3-8], and
Fig 1) Common drug delivery system model. The antibody (purple) targets
drug carriers (yellow) to a specific site within the body. This study establishes
a standard method for linking (red) targeting molecules onto
buckminsterfullerenes, which have shown great potential as drug carriers.
Targeting molecule
Drug
Drug carrier

4. 4
unique abilities to encapsulate drugs within their structures [9-12] and release toxic reactive
oxygen species (ROS) under specific wavelengths of light [2,13,14] make fullerenes highly
efficient and versatile drug carriers with distinct advantages over currently available carriers such
as liposomes and gels as well as other nanoparticle carriers.
A prerequisite for research into this area is the development of chemical methods to
immobilize targeting molecules onto fullerene surfaces in a reliable manner. Thus far, only a
single study has been carried out in this regard. The antibody ZME-018 was successfully
immobilized onto buckminsterfullerene C60 [8]; however, this work does not provide for simple
and broad-range immobilization of targeting molecules onto fullerene surfaces. As a result,
buckminsterfullerenes have not seen much application into targeted drug delivery.
The purpose of this study is to develop a simple method to immobilize a wide range of
targeting molecules onto fullerene surfaces. This will mark a revolutionary progression in
targeted drug delivery, as it will bridge the gap between advances in targeting molecule
development and drug carrier development and allow for a variety of treatments to be assembled
using the exact same, simple production mechanism.
Cytotoxins
Radioactive particles
Therapeutic fullerene
Immobilization
Virally-infected cells
Bacterial cells
Cardiac tissue
Cancers
Targeting
Pharmaceuticals Targeting Molecules Targets
Fig 2) Model for targeted therapeutic system using fullerene drug carriers. A wide range of
pharmaceuticals have been attached onto or within fullerene surfaces, and targeting molecules
have been developed against a number of disease-specific biomarkers. However, no general
method currently exists to immobilize targeting molecules onto fullerene surfaces (red). Such a
method would allow for a simple, universal method for the assembly of targeted drug delivery
systems, removing problems of complex/inefficient production that currently prevent these
systems from affordability and reliability.

5. 5
Materials and Methods
The goal of this study is to develop a simple and general method for immobilization of
functional targeting molecules onto buckminsterfullerene surfaces. Fullerenes are unique among
solid-state materials in that every atom is on the surface, indicating that surface chemistry could
be critical to their physical properties and applications [22]. It is therefore imperative that any
immobilization methodology preserves the structure of these molecules and thus their physical
and chemical characteristics. Targeting molecule functionality is also heavily dependent on
molecular structure, making its preservation imperative as well [18-21]. This study employs an
immobilization method that preserves molecular structures by noncovalently acting upon
fullerenes and acting upon structurally insignificant amine (NH2) tails that are either already
present on molecules (e.g. proteins) or are synthetically added via a simple and efficient process.
At the center of this process is the bifunctional molecule 1-pyrenebutanoic acid,
succinimidyl ester (PASE; Sigma Aldrich, USA), which adsorbs onto the inherently hydrophobic
surfaces of fullerenes in an organic solvent (e.g. dimethylformamide).
The pyrenyl group of PASE, which is highly aromatic in nature, is known to interact
strongly with the basal plane of graphite via π-stacking [23,24], and has also been found to
strongly interact with the sidewalls of single-walled carbon nanotubes (SWNTs) [22]. π-stacking
strength onto buckminsterfullerenes is governed by the isolated pentagon rule (IPR), which
dictates that the most stable interactions occur on fullerenes whose carbon networks contain
isolated pentagons; i.e. all pentagons are surrounded by five hexagons, including fullerene C60
and all fullerenes C70 or larger [25]. Since these are the most commonly used fullerenes with
Fig 3) Structure of 1-pyrenebutanoic acid,
succinimidyl ester (PASE). The pyrenyl group (four
aromatic rings) of PASE undergoes π-stacking
interactions with buckminsterfullerenes;
immobilizing the molecules onto fullerene surfaces.
The N-Hydroxysuccinimide-ester component (right
of the ester) forms amide bonds between PASE and
any molecule with an amine tail, including proteins,
peptides, and modified synthetic molecules. This
presents a general approach for immobilization of
functional molecules onto fullerene surfaces.
Pyrenyl group
NHS-ester

6. 6
regards to applicability [25], π-stacking provides a promising methodology for noncovalent
buckminsterfullerene functionalization.
The mechanism of molecular immobilization onto buckminsterfullerenes involves
nucleophilic substitution of N-Hydroxysuccinimide from PASE by an amine group on the
targeting molecule, resulting in the formation of an amide bond [22]. This enables the
immobilization of a wide range of targeting molecules onto buckminsterfullerene surfaces.
Fig 4) Examples of
targeting molecules that can
be immobilized onto
buckminsterfullerenes
through mediation by 1-
pyrenebutanoic acid,
succinimidyl ester,
including proteins (upper
left) [33], oligonucleotides
(upper right) [34], and small
molecules (bottom).
Inset: Visualization of
fullerene-PASE π-stacking.
Targeting molecule
Pyrenel moiety of PASE + C60
R T R
T
R
T
T
H2O (Hydrolysis)

7. 7
Functionalization Procedure
In this study, aptamer (oligonucleotide) AS1411 was immobilized onto fullerene C60
through mediation by 1-pyrenebutanoic acid, succinimidyl ester. An aptamer was used in this
study due to relative simplicity in handling as well as proof-of-concept that the methodology
used in this study is applicable to immobilization of synthetic molecules onto fullerene surfaces,
as PASE-protein linking has already been well established in literature [22].
Buckminsterfullerene C60 (Sigma Aldrich, USA) was suspended in the water-miscible
organic solvent dimethylformamide (DMF; Sigma Aldrich, USA) at 5 mg/mL. 1-pyrenebutanoic
acid, succinimidyl ester (Sigma Aldrich, USA) was dissolved in tetrahydrofuran (THF; Sigma
Aldrich, USA) – an organic solvent miscible with DMF and water and capable of efficiently
dissolving PASE – at 5 mg/mL. The two mixtures were incubated with stirring (400 rpm) at
Fig 5) Schematic of amide bond formation between N-Hydroxysuccinimide ester and
primary amine tails. Competitive hydrolysis of esters can harm efficiency of amide bond
formation; however, this reaction is highly inefficient relative to that of NHS and primary
amines without catalysts [26].
Fig 6) Schematic for immobilization of aptamer AS1411 onto buckminsterfullerene C60.

8. 8
room temperature for 2 hours. Following incubation, the mixture contained C60 functionalized
with PASE and potentially unreacted C60 and PASE.
Amine-modified aptamer AS1411 (5’–NH2–C6–AS1411; GenScript, USA) was dissolved
in 0.02 M phosphate buffer saline (PBS, pH 7.2) at 2 mg/mL. This solution was incubated with
the C60/PASE/DMF/THF mixture from the last step at 4°C with shaking for 18 hours.
Functionalized C60 was purified via liquid-liquid extraction. Toluene is an efficient
solvent of C60 and PASE and is immiscible in water, THF, and DMF. Addition of toluene,
shaking, then removal of toluene layer constituted one wash, which removed excess C60, PASE,
N-Hydroxysuccinimide byproduct from PASE-amine reaction, and C60 functionalized with PASE
that did not bind to aptamer. Thirty washes were performed in total.
The final mixture was frozen in liquid nitrogen then placed in a sublimation vacuum,
which removed all solvent from the final product.
Characterization
Success of functionalization was tested via attenuated total reflectance–Fourier transform
infrared (ATR-FTIR) spectrometry. ATR-FTIR is a popular qualitative characterization
technique used to detect presence of certain chemical groups within molecules; it is used in this
study to detect presence of C60, PASE, and AS1411, indicating successful immobilization.
An attenuated total reflection accessory operates by measuring changes that occur in a
totally internally reflected infrared beam when this beam comes into contact with a sample,
generating an infrared (IR) spectrum specific to the sample chemical. Each peak within an IR
spectrum represents a vibrational mode within a certain chemical bond or group, such as
symmetric/antisymmertric stretching, scissoring, rocking, wagging, and twisting.
***Note: All procedures were performed by author except for: handling of liquid nitrogen and
related protocols; handling of sublimation vacuum and related protocols. These procedures were
performed courtesy of properly trained graduate students. Proper training was provided for the
use of all other materials and protocols by laboratory personnel prior to the beginning of the
study.***

9. 9
Results
Chemical groups and bonds within C60 and 1-pyrenebutanoic acid, succinimidyl ester
have certain responses at specific wavelengths of light within the range 2000 cm-1
to 600 cm-1
.
These responses are displayed in the IR spectrum as downward peaks. Most nucleic acid
structures, including aptamers, contain phosphates as their sole chemical group that can be read
within the specified IR range (2000 cm-1
to 600 cm-1
). However, presence of a phosphate merely
indicates aptamer presence within the media, and does not indicate whether or not the aptamer
successfully immobilized onto the buckminsterfullerene via PASE. Thus, successful aptamer
immobilization was determined via indicators of amide formation with the ester group on PASE,
i.e. absence of C=O stretching vibration of N-Hydroxysuccinimide, a byproduct of the NHS ester
– amine reaction that is removed via toluene washing, instead of peaks corresponding to
vibrational modes of chemical groups on the molecule itself.
%Transmittance
Wavenumber (cm-1
)
C=O of ester in PASE
C
60
C
60
C=O of N-
Hydroxysucci
nimide in
PASE,
washed out
as by-product
PO4 of aptamer
Fig 7a) ATR-FTIR spectrum of C60-PASE-AS1411 construct.

10. 10
C60
C60 contains two major vibrational modes along with a number of smaller modes within
the range 2000 cm-1
to 600 cm-1
that are identifiable via infrared spectroscopy. These modes vary
slightly in wavenumber, as they represent vibrations of the entire C60 molecule and are affected
by C60 aggregation and incorporation of new molecules onto or near the C60 structure [27]. Thus,
IR results are considered valid so long as they fall within 5% of theoretical results [28]. The first
major mode, termed T1u(3), lies at ~1182 cm-1
based on prior experimental results [27] and 1218
cm-1
based on theoretical results [28]. The second, termed T1u(4), lies at ~1429 cm-1
based on
prior experimental results [27] and 1462 cm-1
based on theoretical results [28]. Both peaks in the
obtained spectrum fall within 5% of theoretical values, indicating presence of C60.
Obtained Theoretical Experimental % Deviation (from Theoretical; Experimental)
1206 cm-1
1218 cm-1
1182 cm-1
0.9950%; 2.030%
1390 cm-1
1462 cm-1
1429 cm-1
4.925%; 2.729%
Fig 7b) Vibrational modes corresponding to peaks in spectrum.
Top row – T1u(3)
Bottom row – T1u(4)

11. 11
AS1411 (Immobilized molecule)
Aptamer AS1411 contains one IR active chemical group – a P=O of phosphate groups in
the molecule – within the given range. P=O experiences stretching vibration, represented by a
strong peak, at 1234 cm-1
[29]. However, this merely indicates presence of aptamer in the
medium, and not necessarily successful immobilization, since excess aptamer is not removed via
toluene washing and the peak could represent both immobilized and not immobilized aptamer.
Success of immobilization is discussed in the next section.
1-pyrenebutanoic acid, succinimidyl ester (PASE)
1-pyrenebutanoic acid, succinimidyl ester contains two chemical groups with vibrational
modes within the given range [30]. The first, a C=O stretching vibration within the ester group of
the molecule, is represented by a strong peak at 1736 cm-1
. The second, a C=O stretching
vibration within the N-Hydroxysuccinimide (NHS) group, is represented by a strong peak at
1774 cm-1
.
This peak is absent from the IR, indicating that NHS is removed from the final product.
This happens through two reaction pathways: the first is conjugation of PASE to an amine group;
Fig 8) Corresponding structures for C60 peaks, 2000 cm-1
– 600 cm-1
. C60 vibrational modes involve
vibrations of the entire C60 molecule rather than specific bonds within the structure, causing deviations
within mode wavenumbers due to C60 aggregates and presence of other molecules (e.g. the pyrenyl
group of PASE) on C60 structures. Obtained values that deviate less than 5% from theoretical values
(Giannozzi et al.) are considered valid; the most recent experimental values (Menéndez et al.) of modes
T1u(3) and T1u(4) are also presented for reference.
Fig 9) Corresponding structures for 1-pyrenebutanoic acid, succinimidyl ester peaks, 2000 cm-1
–
600 cm-1
.
C= O stretching vibration;
1774 cm-1
C= O stretching vibration;
1736 cm-1

12. 12
the second is via competitive (albeit inefficient due to lack of catalyst) hydrolysis of the ester
group on PASE. The products of hydrolysis are removed via toluene washing; therefore, only the
IR active groups on PASE that remain following amine conjugation are displayed on the
spectrum. It is thus reasonable to infer that the ester group indicated in the spectrum belongs to
PASE successfully reacted with the targeting molecule (aptamer AS1411).
Presence of C60 in the spectrum indicates successful π-stacking with PASE. Overall, the
collective presence of all the mentioned peaks indicates successful immobilization of the
targeting molecule, aptamer AS1411, to C60.
Discussion
These findings represent the first successful noncovalent immobilization of functional
molecules onto a buckminsterfullerene surface, opening doors to a universal scaffold for targeted
drug delivery system assembly.
Fullerenes have attracted considerable attention in medical applications due to their
appealing photochemical, electrochemical, and physical properties [3]. Studies into the medical
potential of fullerenes have primarily been divided into: cytotoxin delivery, photodynamic
therapy, and immunoradiotherapy.
Fig 10) Corresponding structures for 1-pyrenebutanoic acid, succinimidyl ester peaks post-
reaction with primary amine on AS1411, 2000 cm-1
– 600 cm-1
.
C= O stretching vibration;
1736 cm-1
C= O stretching vibration;
1774 cm-1
; not present in IR
(washed with toluene)

13. 13
Zakharian et al. have successfully covalently conjugated fullerene C60 to the
chemotherapeutic drug paclitaxel [5]. The conjugate released paclitaxel via enzymatic hydrolysis
and subsequently demonstrated half-life of release of 80 minutes in bovine plasma and
significant cytotoxicity in tissue culture, indicating promise for increased therapeutic efficacy of
paclitaxel in vivo. In a later study, Ashcroft et al. conjugated fullerene C60 to anti-melanoma
antibody ZME-018, establishing the first method for targeted (site-specific) drug delivery via
fullerenes [8]. However, fullerene-biomolecule conjugation as per the method established by
Ashcroft et al. is highly complex, and as a result, highly inefficient.
The work presented in this study indicates a much simpler, two-step method for
immobilization of targeting molecules, such as antibodies, onto fullerene surfaces. Further
affinity chromatography studies need to be completed in order to determine reaction efficiencies;
however, it is expected that the presented methodology will have significantly higher efficiency
than that of Ashcroft et al. due to the smaller number of reactions and the high efficiency of
pyrenyl π-stacking onto fullerene surfaces. In addition, the presented methodology does not
interfere with either fullerene or target molecule structure, whereas that of Ashcroft stretches C60
structure. The versatility of this work also allows for protein conjugation without modification
(the Ashcroft method required that the protein be modified with thiol groups), and is geared
toward immobilization of a wide array of targeting molecules, including synthetics, onto
fullerene surfaces (the Ashcroft method was designed solely for immobilization of a limited
group of antibodies onto fullerene surface).
Fullerenes have also been conjugated to oligonucleotides for gene therapy – Yang et al.
have demonstrated that fullerene C60 conjugated to oligonucleotide sequence complementary to a
specific region of β-actin cDNA not only inhibited Taq DNA polymerase and the cDNA
template, but also inhibited the activity of exonuclease I due to the protein’s affinity to C60 [15].
This method did not provide for targeting of fullerene molecules to specific cells, however. The
findings from the our study support a simple method of immobilization of targeting molecules
onto fullerene surfaces, significantly increasing therapeutic efficiency, thus increasing the
applicability of buckminsterfullerenes in gene therapy. Fullerenes are also able to cross the
highly selective blood-brain barrier (BBB), indicating promise of the molecules in neural
therapies as well [3]. Lastly, the presented method provides for simple immobilization of
synthetic molecules such as aptamers and small molecules onto buckminsterfullerene surfaces

14. 14
without altering the structure, and therefore the functionality, of any molecule, opening doors to
purely synthetic drug delivery systems, which are very low cost and easy to manufacture
compared to their biological counterparts (e.g. antibody-based systems). Thus, PASE-
functionalized buckminsterfullerenes represent a highly promising general scaffold for a wide
variety of targeting molecules such as small molecules, aptamers/oligonucleotides, and
peptides/proteins. This allows for assembly of targeted drug delivery systems against an
immense range of diseases through the exact same, simple, cost-efficient methodology.
Fullerenes have also shown great promise as photosensitizers in photodynamic therapy
(PDT) – a highly promising, noninvasive, light-activated cancer treatment – due to their high
photostability, photochemical versatility (fullerene produce singlet oxygen, hydroxyl radicals,
and superoxide anion upon illumination; these reactive species serve as effective PDT
mediators), and ease of modification [2,13,14]. Fullerenes have not seen application as
photosensitizers, however, in large part due to their inability to be targeted specifically to cancer
cells. Similar to targeted drug delivery, the methodology provided in this study can be used to
immobilize targeting molecules onto fullerene, thereby increasing efficiency, and therefore
applicability, of buckminsterfullerenes in PDT.
Metallofullerenes – fullerenes with metallic particles enclosed within their structures –
have shown great promise in radiological applications. Shultz et al. have described the use of
theranostic metallofullerenes for imaging (f-Gd3N@C80) and treatment (177
Lu-DOTA-f-
Gd3N@C80) of gliomas [10]. More recently, Diener et al. found that 212
Pb@C60 demonstrated
Fig 11) Schematic for a fullerene-based
targeted drug delivery system loaded
with cytotoxins (colored). Note that one
fullerene molecule can carry multiple
drugs, allowing for a greater number of
drugs to be delivered per targeting
molecule. This also allows for drug
“cocktails”, or the administration of
different drugs, each with a unique
therapeutic purpose (e.g. a
chemosensitizer and cytotoxin), within
the same treatment, allowing for highly
efficient therapies.

15. 15
stable radioactive decay and did not accumulate in bone upon administration in vivo as a novel
radioimmunotherapy (RIT; targeted radiation therapy) agent [11]. Berger et al. have successfully
internalized gadofullerene-antibody conjugates (Gd@C60 – ZME-018) into melanoma cells;
however, the method used to immobilize the antibody onto fullerene surfaces (Ashcroft method,
described prior) was highly inefficient and did not provide for immobilization of a wide range of
other targeting molecules [12]. Nonetheless, Berger et al. demonstrated highly promising results
as to the practicality of fullerenes in radiotherapeutics. This, coupled with the simple
immobilization of targeting molecules onto fullerene surfaces presented in this study, provides
for a highly promising radioimmunotherapeutic that avoids all major problems of current RIT;
i.e. accumulation in bone, unreliable conjugation between targeting molecules and radioactive
isotopes, and unstable decay of these isotopes.
Conclusion
In summary, this study presents a controlled method for immobilizing a wide array of
functional molecules onto fullerene surfaces, providing the foundation for a simple and general
method for the assembly of targeted drug delivery systems.
In this study, aptamer (oligonucleotide) AS1411 was immobilized onto
buckminsterfullerene C60. Immobilization was proven successful via infrared spectrometry;
however, further chromatography studies are required in order to assess reaction efficiencies.
Fig 12) Schematic for a
fullerene-based targeted drug
delivery system loaded with a
radioactive particle (red).

16. 16
This study utilized pure C60, which is water insoluble by nature, for sake of simplicity.
While insoluble C60 is acceptable, and in many cases advantageous, for some delivery
applications, most therapeutic fullerenes must show water solubility in order to successfully be
implemented as medicinal systems. Future work will therefore focus on molecular
immobilization onto water-soluble fullerenes. It is expected that the methodology will stay
largely the same; the only expected differences are incorporation of water in place of organic
solvents and use of N-(1-pyrenebutanoyl)cysteic acid, a water-soluble bifunctional molecule with
the same major structural features as 1-pyrenebutanoic acid, succinimidyl ester [31], in place of
PASE.
The methodology used in this study is applicable to nearly any combination of fullerene
and drug; since the structure of neither component is affected during production, the structural
integrity, and therefore efficacy, of any assembled system is always upheld.
Lastly, the presented methodology is very easy to implement, allowing for simple
incorporation into nearly any present fullerene-based therapeutic system under development,
thereby providing an invaluable tool toward the progression of targeted drug delivery systems,
and consequently the assembly of safer, more efficient treatments for a host of diseases using one
simple, standard method.
***Note: All images, tables, and graphs were created by the author. 3-Dimensional
visualizations were created with PyMOL molecular visualization software [32].***

17. 17
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