A Simple Two-Step Method for Molecular Immobilization Onto Buckminsterfullerenes

Sairaj Sajjath

1/1/2015

  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  

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

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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.

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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

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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  

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  

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.***

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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.

% T

rans

mitt

ance

Wavenumber (cm-1)

C=O of ester in PASE

C60

C60

 

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.

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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)

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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

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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  

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  

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 (177Lu-DOTA-f-

Gd3N@C80) of gliomas [10]. More recently, Diener et al. found that 212Pb@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  

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  

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  

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