A Comparative Study of Two Folate-Conjugated Gold Nanoparticles ...

Cancers 2010, 2, 1911-1928; doi:10.3390/cancers2041911
OPEN ACCESS
cancers
ISSN 2072-6694
www.mdpi.com/journal/cancers
Review
A Comparative Study of Two Folate-Conjugated Gold
Nanoparticles for Cancer Nanotechnology Applications
G. Ali Mansoori 1,*, Kenneth S. Brandenburg 1 and Ali Shakeri-Zadeh 2
1 Department of Bioengineering, University of Illinois at Chicago, 851 S. Morgan St. (MC 063),
Chicago, IL 60607, USA; E-Mail: kbrand@uic.edu
2 Department of Medical Physics, Tehran University of Medical Sciences, Tehran, Iran;
E-Mail: shakeri2005@gmail.com
* Author to whom correspondence should be addressed; E-Mail: mansoori@uic.edu;
Tel.: +1-312-996-5592.
Received: 29 October 2010; in revised form: 10 November 2010 / Accepted: 11 November 2010 /
Published: 18 November 2010

Abstract: We report a comparative study of synthesis, characteristics and in vitro tests of
two folate-conjugated gold nanoparticles (AuNP) differing in linkers and AuNP sizes for
selective targeting of folate-receptor positive cancerous cells. The linkers chosen were
4-aminothiophenol (4Atp) and 6-mercapto-1-hexanol (MH) with nanoconjugate products
named Folate-4Atp-AuNP and Folate-MH-AuNP. We report the folate-receptor tissue
distribution and its endocytosis for targeted nanotechnology. Comparison of the two
nanoconjugates’ syntheses and characterization is also reported, including materials and
methods of synthesis, UV-visible absorption spectroscopic measurements, Fourier
Transform Infra Red (FTIR) measurements, Transmission electron microscopy (TEM)
images and size distributions, X-ray diffraction data, elemental analyses and chemical
stability comparison. In addition to the analytical characterization of the nanoconjugates,
the cell lethality was measured in HeLa (high level of folate receptor expression) and
MCF-7 (low level of folate receptor expression) cells. The nanoconjugates themselves, as
well as the intense pulsed light (IPL) were not harmful to cell viability. However, upon
stimulation of the folate targeted nanoconjugates with the IPL, ~98% cell killing was found
in HeLa cells and only ~9% in MCF-7 cells after four hours incubation with the
nanoconjugate. This demonstrates that folate targeting is effective in selecting for specific
cell populations. Considering the various comparisons made, we conclude that

Folate-4Atp-AuNP is superior to Folate-MH-AuNP for cancer therapy.

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Keywords: 4-aminothiophenol; 6-mercapto-1-hexanol; cancer nanotechnology; folate;
folate receptor; folic acid; gold nanoparticle; nanoconjugate; photothermal treatment

1. Background and Introduction
A need exists to target cancer treatments specifically to the tumor site, without damaging healthy
tissue. The answer to solving this challenge lies in the successful application of nanotechnology to
cancer treatment [1]. Nanotechnology is based on the 1–100 nanometer scale as its name implies [2,3].
Since nanotechnology exists well below the size of the cell (10,000–100,000 nm), it is a great
candidate for solving such problems. Due to its size, nanotechnology offers the potential to selectively
seek out and destroy cancerous tissues by a variety of targeting and destruction methods.
A malignant cancer is typically fast growing, which means that it requires more nutrients and
increased waste removal than healthy tissues. Nanoparticles injected into the bloodstream can enter
into tumors because of the defects or pores within the tumor vasculature. However, the residence time
within the tumor is limited due to random diffusion into and out of the tumor. Therefore, increasing the
residence time of nanotechnology-based cancer treatments within tumors is necessary. To achieve
increased residence time, active targeting must be used. Active targeting is based on selectively
targeting cancer cells through a specific binding site on the surface of the cell, such as a receptor.
Various methods of targeting cancer cells have been proposed [1,4]. One promising targeting method
is with folic acid or folate, the folic acid salt, which is the subject of the present report.
Folic acid is also named pteroylglutamic acid and has the closed chemical formula C19H19N7O6
(Mw = 441.4 Da) and open chemical structure as shown in Figure 1.
Figure 1. The molecular structure of folic acid.




Folic acid or folate (pteroylglutamate) is water-soluble and is brought into both healthy and cancerous
cells by the folate-receptor. This receptor is used to transport folate into the cytosol for the synthesis of
thymine by dihydrofolate reductase. The presence of the folate-receptor on a cell’s surface is regulated
by the cell’s function. Cancer cells tend to overexpress the folate-receptor because of their vast
requirement for folate. It has been proposed that the folate-receptor makes for a suitable targeting agent
because of its relatively low expression level in healthy tissues and overexpression in cancerous tissues.
A positive aspect of folate is its possible conjugation with a number of nanotechnology platforms,
such as gold nanoparticles and chemotherapeutic agents. When these nanotechnology platforms are
deposited at the tumor site a variety of methods to eradicate the cancer cells can be used, such as
thermal ablation, drug release or delivery, or even coating the cancer cells with a high affinity antigen,

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which the body’s immune system can detect and mount a defense against. Due to folate’s promising
characteristics of non-immunogenic, specificity for cancer, and its possible conjugation with gold
nanoparticles (AuNP) as reported here, folate-AuNP nanoconjugate is a front-runner as a targeting
moiety for many cancer nanotechnology treatments.
1.1. Folate-Receptor Tissue Distribution
The folate-receptor is a glycosyl-phosphatidylinositol linked membrane protein with a molecular
weight of 38,000 Daltons. Immunocytological tests have shown the presence of folate-receptor in the
ovaries, kidneys, lungs, thyroid, the fallopian tube, as well as several ovarian cancers. According to
Weitman and Kamen [5], the folate-receptor is commonly expressed within several healthy tissues of
the body. Among the most positive tissues that express the folate-receptor are the choroid plexus,
kidney, and the lungs. If these healthy tissues commonly express the folate-receptor then how would a
cancer treatment targeting the folate-receptor be effective? The answer lies in the biology of the
vasculature of healthy and cancerous tissue as well as the membrane localization of the folate-receptor.
The most important part of this answer is the membrane localization. Epithelial cells, those forming a
layer separating the body’s tissues from either the outside (skin) or from the interior cavities such as
the gastrointestinal tract or lungs, have two distinct membrane forms. One (the basal membrane) is
facing the other tissues or the bloodstream and the other (apical membrane or luminal surface) is
facing the outside or cavity within the body. The folate-receptors within healthy tissues are localized
on the apical membrane of the epithelial cells. This means that the folate-receptors in the choroid
plexus are entirely expressed toward the cerebrospinal fluid. Likewise, for the kidney and lungs the
folate-receptors are expressed toward the urine and air, respectively [6]. This was also found to be true
in the gastrointestinal tract.
The secondary part of the answer to the original question is in regard to the vasculature differences
between the healthy and cancerous tissues. As mentioned above, the vasculature of tumors is filled
with defects, which are in the hundreds of nm size range, whereas the pores in the healthy tissue
vasculature are in the few nanometer size range. This means that any targeted nanotechnology
treatment agent must be larger than 10 nm to prevent the particle from entering healthy tissue. Also,
the treatment agent must be smaller than 100 nm to allow it to enter the tumor. Therefore, the size of
the nanoparticle treatment agent should prevent it from accessing the folate-receptor in healthy tissue.
This may not apply if the patient has tissue damage, where the vasculature is damaged, or has been
exposed to permeability enhancers, such that pore size is increased in healthy tissues. Since the
nanoparticle treatment agent will be able to diffuse into the tumor through the defects in the tumor
vasculature, the particles will only be exposed to the folate-receptor on the cancer cells [7].
It was shown that certain cancer cells overexpress the folate-receptor on an order of 100-times more
than normal healthy cells [8]. This overexpression of folate-receptor in the cancer cells makes folate a
suitable targeting agent for such cancer cells. It should be noted that malignant cancer, which continues
growing into the surrounding tissues, would not only express a high number of folate receptors on its
surface but also has high folate-receptor. The limited and well localized tissue distribution of the
folate-receptor within healthy tissues, the defects in tumor vasculature, and the high expression level
within several types of cancer make folate an appropriate choice of targeting moiety for
nanotechnology based cancer treatment.

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1.2. Folate-Receptor Endocytosis for Targeted Nanotechnology
Understanding the molecular mechanisms of the folate-receptor endocytosis within tumors allows
for the selective targeting of cancer cells [8]. In humans, the normal blood concentration of folate is
approximately 6.8–36 (nanomol/L (3–16 nanogram/mL) and is regulated by the kidney [6]. It has been
estimated that 400 µg of folate must be replaced by dietary intake every day [9]. When folate is
consumed the blood concentration will normally increase dramatically, and will almost immediately
begin to be filtered out by the kidneys. Before the kidneys filter the excess folate, the cells of the body
can uptake it if folate can bind to the cells’ folate-receptors.
Folate has a strong binding affinity for its receptor. Folate has an association constant of
KA = 2 × 107 [s], i.e.,
[Folate - Receptor 
Aggregate]

Folate

K 
 2 107
x
[s]

(1)
A


[Folate -

[Folate]

Receptor]


as reported by Hong et al. [10]. The association constant describes the bonding affinity between folate
and its receptor at equilibrium. The binding and association of folate is important when designing the
nanoparticle construct that targets the folate-receptor.
We know the vasculature within tumors contain many defects, which allow particle sizes around
and below 100 nm to be passively deposited at tumor sites [11-14]. Passive targeting allows for
increased depositing of nano-carriers within the tumor but does not guarantee their cellular uptake [15].
When the nano-carrier is passively targeted to tumors, it can remain within the tumor or it can also
diffuse out of the tumor and back into the bloodstream, due to the high interstitial pressure within solid
tumors and random diffusion [16]. Therefore, in order for the nano-carriers to achieve a greater affinity
to, and residency time in, the tumor, as well as enter the cancer cells themselves, active targeting must
be employed. By utilizing the cancer cells own deficiencies, need for folate, against themselves various
nanoparticles can be used to target cancer cells [1,4,17-20]. In this project we choose the gold
nanoparticle due to its unique properties. Folate can be conjugated to gold nanoparticles
(nanoconjugate) through a linker as shown in Figure 2.
Figure 2. Schematics of a nanoconjugate of gold nanoparticle with folate (AuNP-Linker-Folate).

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The nanoconjugate is internalized by the folate-receptor (see Figure 3) in an endocytotic pathway,
which does not enter into a clathrin coated pit pathway. This means that the nanoconjugate is released
directly into the cytosol rather than being transported to an endosome or lysosome by intracellular
vesicle transport.
Figure 3. Various stages of (AuNP-Linker-Folate) nanoconjugate transfer in the cell
through the folate-receptor on the cancer cell’s membrane.

The folate-receptor is linked in the lipid region of the membrane allowing it the ability to migrate
through the membrane to release its content into the cytosol. Each caveolae is estimated as having
approximately 750 folate-receptors in it, giving an average receptor density of 32,000/m2 on each
cancer cell. Figure 3 shows the caveolae beginning to close after the folate binds to the folate-receptor.
The closed caveolae will begin to migrate to the interior surface of the phospholipid bi-layer. During
this migration, the interior of the caveolae becomes more acidic reaching a pH of approximately 5,
which causes dissociation of the (AuNP-Linker-Folate) nanoconjugate from the folate-receptor. The
nanoconjugate is then released into the cytosol of the cancer cell upon reaching the interior surface of
the cell membrane. Once the nanoparticles are internalized by the cancer cells, the cytotoxic agents can
be released or in the case of gold nanoparticles, thermally ablate the cancer cells.
Important aspects to notice in this endocytotic pathway are the acidification of the interior of the
caveolae during migration, the size of the caveolae, the absence of forming a vesicle for intracellular
transport, and the dumping of the nanoconjugate directly into the cytosol. All of these features can be
utilized in the final design scheme of a nanotechnology-based treatment of cancer.
Due to its possibility of conjugation, non-immunogenic properties, and requirement for cancer cell
growth, folate is a novel targeting agent for malignant tumors. For the in vivo stage of our proposed
work we may use different shaped and sized AuNPs including the advanced Au nanorods [21] and Au
nanoshells [22]. AuNPs heat up as a result of absorption of visible light, which makes them suitable for
use in cancer photothermal treatment.

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1.3. Nanoconjugates Synthesis and Characterization
We developed and reported the original bioengineering design of this cancer nanotechnology
process in 2005 and we reported our results in early 2006 [23]. Simultaneously, we developed a
biosynthesis method for large scale production of metallic nanoparticles [24]. As a result of our
research we have completed six research projects [25-30]. Meanwhile, two related papers by other
groups [20,21] have reported on other folate-AuNP nanoconjugates. Here we report and compare two
different folate-AuNP nanoconjugates that we have produced and tested. This includes comparison of
their synthesis, characteristics and in vitro tests. The two nanoconjugates are named Folate-4Atp-AuNP
and Folate-MH-AuNP, which are the results of conjugation of AuNP with folate by 4-aminothiophenol
(4Atp) and 6-mercapto-1-hexanol (MH), respectively.
2. Materials and Methods
The reagent grade chemical and biological compounds that are used in this research are as follows:
Hydrogen tetrachloroaurate (III) trihydrate (HAuCl .43H2O), 4-aminothiophenol (C6H7NS), 6-mercapto-
1-hexanol (C
), N, N'-dicyclohexylcarbodiimide (
),
6H14OS), Sodium borohydride (NaBH4
C13H22N2
Folic acid (C
), Trypan blue, RPMI 1640, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-
19H19N7O6
diphenyltetrazoliumbromide), dimethylsulfoxide (DMSO), streptomycin, penicillin and trypsin-EDTA,
Fetal calf serum (FCS), and HeLa and MCF7 cell lines.
The details of the reagents’ commercial makers, cell culture procedure, synthesis and preparation of
Folate-4Atp-AuNP and Folate-MH-AuNP nanoconjugates are already reported [25-30]. In Figure 4,
schemes for the synthetic procedures of the two nanocongugates are shown on a comparative basis.
The final products of the synthesis are Folate-4Atp-AuNP nanoconjugate in powder form,
exhibiting a golden color in reflection, and Folate-MH-AuNP nanoconjugate, also in powder form,
with a deep brown color. The synthesis powder results were stored for further characterization and
in vitro tests as described below.
Figure 4. Schemes for the synthetic procedures of folate conjugated with AuNP using
4Atp (left scheme) [25-27] and MH (right scheme) [28,29] as the linkers to produce
nanocongugates.

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2.1. Characterization of Nanoconjugates
For characterization of nanoconjugates, the following tests were performed: (i) UV-visible
(UV-vis) absorption spectroscopy; (ii) Fourier Transform Infra Red (FTIR) measurements;

(iii) Transmission electron microscopy (TEM); (iv) X-ray diffraction (XRD) and; (v) Elemental
analyses. Details of these techniques and analysis were reported earlier [25-30]. The comparative
results of the two nanoconjugates are reported blow.
2.1.1. UV-Vis Spectroscopy
The UV-visible absorption spectroscopic measurements were recorded on a single beam UV-vis
spectrometer, Agilent 8453, using quartz cells of 1 cm path length and methanol as the reference
solvent at room temperature. It is known that AuNPs possess the characteristic surface plasmon
absorption at 520 nm in the UV-visible absorption spectrum. This characteristic absorption band in the
assemblies of AuNPs interlinked by various molecules may shift exhibiting a peak between 520 and
620 nm. Because of the propensity for intermolecular hydrogen bonding in the assemblies of AuNPs
interlinked by different ligands, resultant broadening and red-shifting of the plasmon absorption peak
are to be expected [24,31,32].
Figure 5. UV-Visible absorption spectra of Folate-4Atp-AuNP [25-27] and

Folate-MH-AuNP [28,29] nanoconjugates.

Figure 5 shows the UV-vis spectra of Folate-4Atp-AuNP and Folate-MH-AuNP nanoconjugates. In
the spectra of both nanoconjugates, the absorption maxima at 280 and the saddle points at 360 nm are
confirmations of the covalent attachment of the folate with Atp-AuNP and MH-AuNP [33]. For the
photothermal ablation of cancer cells reported below, a Lumenis intense pulsed light (IPL) source was
used. Since the absorption peaks at ~560 nm in both nanoconjugates pertain to AuNP then the 560 nm
filter of IPL source for irradiating of samples were found appropriate. According to Figure 5, the
absorption peak of Folate-4Atp-AuNP is much sharper than that of Folate-MH-AuNP, but the level of
absorption for both at ~560 nm are identical.

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2.1.2. Fourier Transform Infra Red Spectroscopy
The Fourier Transform Infra Red measurements of the two nanoconjugates were recorded on a
Shimadzu FT-IR 4300 instrument using KBr pellets at room temperature. Figure 6 shows the FTIR
spectra of Folate-4Atp-AuNP and Folate-MH-AuNP.
Figure 6. Fourier Transform Infra Red (FTIR) spectra of Folate-4Atp-AuNP [25-27] and
Folate-MH-AuNP [28,29].

According to this Figure, the FTIR spectra of Folate-4Atp-AuNP and Folate-MH-AuNP show the
carbonyl absorbance at 1,700 cm-1 due to (–CONH–) and (–COO–) groups, respectively. However the
–CONH– absorbance seems a bit stronger than the (–COO–) absorbance. In addition, the bands
between 3000–3700 of both conjugates belong to the amine (–NH2) and amide (–CO–NH–) stretches
of folate. Additionally, the bands below 1700 correspond to the out-of plane and in plane motions of
(–NH2) and (C–N=) stretches of folic acid.
2.1.3. Transmission Electron Microscopy
Transmission electron microscopic images of the nanoparticles were taken with a LEO 912AB
instrument operated at an accelerating voltage of 120 kV with line resolution of 0.3 nm at room
temperature. The samples for TEM measurements were prepared by placing a droplet of the colloidal
solution onto a carbon-coated copper grid and allowing it to dry in air naturally. Based on the TEM
images as shown in Figure 7, the size distributions of the final product were determined by counting at
least 300 particles shown as insets in Figure 7.
According to Figure 7, the shapes of nanoparticles are quite spherical and the size histograms
indicate the formation of less polydispersed nanoparticles for Folate-4Atp-AuNP with an average
diameter of 4.8 nm than for Folate-MH-AuNP with an average diameter of about 3 nm.

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Figure 7. Transmission electron microscopy (TEM) photograph of Au nanoparticles in the
two nanocongugates (Left: Folate-4Atp-AuNP; Right: Folate-MH-AuNP). Insets:
histograms for the size distribution of the Au nanoparticles [25-29].

2.1.4. X-Ray Diffraction
X-ray diffraction was carried out with the Bruker D8 ADVANCE X-ray Diffractometer, using the
wavelength of 0.15406 nm (CuKα) radiations at room temperature. As shown in Figure 8, the
crystalline nature of these nanoparticles is confirmed through X-ray diffraction (XRD) analyses.
Figure 8. X-ray diffraction (XRD) patterns of Au nanoparticles in

Folate-4Atp-AuNP (left panel) [25-27] and in Folate-MH-AuNP (right panel) [28,29].



In the case of the Folate-4Atp-AuNP nanoconjugate, as shown in Figure 8, the Miller indices are
(111), (200), (220), (311), (222) and the lattice constants are found to be a = b = c = 0.407376 nm. This
structure is identified as the face-centered cubic (fcc) structure [25]. In the case of Folate-MH-AuNP
nanoconjugate, as shown in Figure 8, the Miller indices are (110), (011), (221), (321), (060), (004) and
the lattice constant is found to be a = 1.348 nm, b = 1.348 nm, and c = 0.725 nm. This structure is
identified as a quasi-cubic structure.

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2.1.5. Elemental Analysis
The elemental analyses for carbon, hydrogen, nitrogen, sulfur and oxygen were performed using a
Thermo Finnigan Flash EA CHNS-O analyzer. The gold percentages in the nanoconjugates were
determined by Shimadzu model AA-670 atomic absorption spectrophotometer.
Elemental analysis of Folate-4Atp-AuNP and Folate-MH-AuNP determined by Carbon Hydrogen
Nitrogen Sulfur Oxygen (CHNS-O) Analysis and Atomic Absorption Spectrometry resulted in the data
reported in Table 1.
Table 1. Elemental analysis data of the nanoconjugates.
Elements
Nanoconjugate 
[Au] [C]
[H] [N] [S] [O] Total [C]:[H] [S]:[H]
Expt’l
[25-27] 41.3 28.7 2.5 13.3 3.5 10.7 99.9 11.48 1.4
Folate-4Atp-AuNP Stochiometric 26.5 40.3 3.1 15 4.3 10.8 100 13
1.39
Expt’l
[28,29] 32 38.2 3.8 11.2 3.6 11.2 100 10.03 0.95
Folate-MH-AuNP
Stochiometric 26.2 39.8 4 13 4.2 12.6 100 9.95 1.05
The gold molecular weight is 196.97. If one folate is conjugated to gold for the two nanoconjugates
their molecular weights would be 744 for Folate-4Atp-AuNP and 753 for Folate-MH-AuNP. This will
amount to 26.5% and 26.2% of gold per nanoconjugate, respectively. Accordingly, the elemental
analysis of the two nanoconjugates indicated that both have one folate conjugated to each gold particle.
The final powder product containing Folate-MH-AuNP has more non-conjugated gold in it than the
powder containing Folate-MH-AuNP. According to this table, the non-conjugated gold content of
Folate-4Atp-AuNP is 9.3% higher than the non-conjugated gold content of Folate-MH-AuNP. The
non-conjugated AuNPs and other chemicals were removed from the systems before the in vitro tests
reported in the next section.
2.1.6. Stability Comparison
The chemical stability of the two nanoconjugates can be compared through the differences in their
bond energies. The only difference in the bonds in formation of the two nanoconjugates are the N–C
bond of (–NH–CO–) between 4-aminothiophenol and folic acid and the O–C bond of (–O–CO–)
between 6-mercapto-1-hexanol and folic acid. The standard bond dissociation energy of the N–C bond
is 73 kcal/mole and that of the O–C bond is 85.5 kcal/mole [34]. This data suggests that the
Folate-MH-AuNP should be more stable than Folate-4Atp-AuNP.
2.2. In Vitro Tests of Nanoconjugates on Cancer Cells
The two nanoconjugates reported above were used to selectively target the folate-receptors that are
overexpressed on the surface of tumor cells. In this section, we report the results of our comparative
study utilizing the two nanoconjugates for the improvement of cellular internalization of AuNP. For
this purpose, human adenocarcinoma HeLa cells were chosen as our model cancer cell line because it
is known to overexpress folate-receptors [35]. HeLa cells are an immortal cell line used in medical
research, which was derived from cervical cancer cells. In addition, MCF7 cell line was selected as the

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