Novel targeted anti-tumor nanoparticles developed from folic acid-modified 2-deoxyglucose

Article abstract of DOI:10.3390/ijms20030697

The glucose analog, 2-deoxyglucose (2-DG), specifically inhibits glycolysis of cancer cells and interferes with the growth of cancer cells. However, the excellent water solubility of 2-DG makes it difficult to be concentrated in tumor cells. In this study, a targeted nano-pharmacosome was developed with folic acid-modified 2-DG (FA-2-DG) by using amino ethanol as a cleavable linker. FA-2-DG was able to self-assemble, forming nano-particles with diameters of 10–30 nm. The biological effects were evaluated with cell viability assays and flow cytometry analysis. Compared with a physical mixture of folic acid and 2-DG, FA-2-DG clearly reduced cell viability and resulted in cell cycle arrest. A computational study involving docking simulation suggested that FA-2-DG can dock into the same receptor as folic acid, thus confirming that the structural modification did not affect the targeting performance. The results indicated that the nano-pharmacosome consisting of FA-2-DG can be used for targeting in a nano-drug delivery system.

Full text of DOI:10.3390/ijms20030697

International Journal of
Molecular Sciences
Article
Novel Targeted Anti-Tumor Nanoparticles Developed
from Folic Acid-Modified 2-Deoxyglucose
Shaoming Jin 1,2 , Zhongyao Du 1, Huiyuan Guo 3, Hao Zhang 3 , Fazheng Ren 1 and
Pengjie Wang 1,3,
*
1
Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science &
Nutritional Engineering, China Agricultural University, Beijing 100083, China; myjackyming@126.com (S.J.);
duzhongyao@cau.edu.cn (Z.D.); renfazheng@cau.edu.cn (F.R.)
2
3
National Institutes for Food and Drug Control, Beijing 100050, China
Beijing Laboratory of Food Quality and Safety, Beijing Higher Institution Engineering Research Center of
Animal Product, Beijing 100083, China; guohuiyuan99@gmail.com (H.G.); zhanghaocau@cau.edu.cn (H.Z.)
Correspondence: wpj1019@cau.edu.cn; Tel.: +86-10-6273-7644
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 12 January 2019; Accepted: 26 January 2019; Published: 6 February 2019
Abstract: The glucose analog, 2-deoxyglucose (2-DG), specifically inhibits glycolysis of cancer cells
and interferes with the growth of cancer cells. However, the excellent water solubility of 2-DG
makes it difficult to be concentrated in tumor cells. In this study, a targeted nano-pharmacosome
was developed with folic acid-modified 2-DG (FA-2-DG) by using amino ethanol as a cleavable
linker. FA-2-DG was able to self-assemble, forming nano-particles with diameters of 10–30 nm.
The biological effects were evaluated with cell viability assays and flow cytometry analysis. Compared
with a physical mixture of folic acid and 2-DG, FA-2-DG clearly reduced cell viability and resulted in
cell cycle arrest. A computational study involving docking simulation suggested that FA-2-DG can
dock into the same receptor as folic acid, thus confirming that the structural modification did not
affect the targeting performance. The results indicated that the nano-pharmacosome consisting of
FA-2-DG can be used for targeting in a nano-drug delivery system.
Keywords: folic acid; 2-Deoxyglucose; nano-particles; targeted anti-tumor; self-assemble
1. Introduction
Cancer has become the disease with the second highest mortality rate worldwide. Currently,
the main therapeutic method for cancer is chemotherapy. However, chemotherapy in cancer treatment
is usually accompanied by adverse effects, since many drugs cannot differentiate between cancerous
cells and normal cells. The targeting of active substances to the tumor region is an effective measure to
solve this problem.
Blocking the consumption of glucose in cancer cells has been shown to be an effective anti-tumor
therapy [
1–
3], because the up-regulation of glycolysis is a near-universal trait in cancer cells that results
in perturbed energy metabolism in cancer cells, in both normoxic and hypoxic environments [
4–6].
Thus, inhibiting the glucose transporters on the cell membrane and the glycolytic enzymes in the
cytoplasm has great potential for killing cancer cells.
The 2-Deoxyglucose (2-DG) is a structural analogue of glucose whose structural difference lies
mainly in the hydroxyl group on the second carbon atom. The 2-DG inhibits glycolysis and reverses
cancer cell metastasis [7–10]. It can inhibit the glucose transporters (GLUTs) on the membranes
of cancer cells because it enters the cytoplasm in the same manner as glucose, via GLUTs [11].
After entering the cell, 2-DG is phosphorylated by hexokinase, the first key enzyme in glycolysis,
forming2-DG-6-phosphate (2-DG-6-P). The 2-DG-6-P is a substrate of phosphoglucose isomerase,
Int. J. Mol. Sci. 2019, 20, 697; doi:10.3390/ijms20030697
www.mdpi.com/journal/ijms

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the second key enzyme in glycolysis, but it cannot be further catalyzed into fructose-6-phosphate
(F-6-P). Thus, the process of glycolysis can be efficiently blocked. The inhibition of hexokinase is
noncompetitive, but the inhibition of phosphoglucose isomerase is competitive [12]. Owing to these
inhibitions, the metabolism of glucose is disturbed, resulting in a decreased energy supply, cell cycle
arrest and inhibited cell viability [13].
However, the excellent water solubility of 2-DG makes it widely distributed in the body. Therefore,
to increase its concentration in tumor cells, targeted transportation of 2-DG to tumors is a necessary
strategy. Highly expressed receptors on the surfaces of cancer cells are a potential target for anti-cancer
therapy [14,15]. The folate receptor, one of these highly expressed receptors, is widely used in anti-tumor
targeting research [16
–
18]. Folic acid, the ligand of the folate receptor, is broadly used in the research and
development of targeted anti-tumor drugs. It can be labeled or loaded with polymers, quantum dots
and nano-materials, for targeting drug delivery. The forms of these nano-scale drug delivery systems
vary considerably, from large polymers with polyethylene glycols [19] to moderate compositions with
metals or quantum dots [20,21] to small nanoparticles with antineoplastic drugs [22,23]. Regardless
of the form of the delivery system, the objective of improving efficacy and reducing toxicity can be
achieved, confirming the advantages of nano-scale drug delivery systems [24,25].
Herein, we designed and synthesized a new drug delivery system consisting of folic acid-modified
2-DG (FA-2-DG). The synthesized small molecule is amphipathic and can self-assemble into
nanoparticles and achieve both passive targeting and active targeting roles. Its biological activity
was evaluated on the basis of cytotoxic effects in vitro in MCF-7 and SKOV-3 tumor cells, and the
related mechanism of action was assessed by evaluating its influence on the cell cycle and its ability to
induce apoptosis.
2. Results and Discussion
2.1. The Synthesis of FA-2-DG
FA-2-DG was synthesized from folic acid and 2-DG, with amino ethanol as a linker agent. Since
the high reaction activity of amino group would introduce by-products and make purification more
difficult, amino ethanol was not directly used in synthetic reactions. First, 1-bromo ethanol was
connected to acetylated 2-DG, and then the bromine atom was replaced by an azide group. In the next
step, an amino group was generated via a reduction reaction catalyzed by a Lindlar catalyst under
a hydrogen atmosphere. The amino and carboxyl groups formed an amide bond. After removal of the
acetyl protection of 2-DG under alkaline conditions, FA-2-DG was synthesized. All structures were
confirmed by 1H-NMR and 13C-NMR. The synthetic route is shown in Figure 1.
OH
OAc
OAc
OAc
OAc
TsO
NH3
p-TSA, H2 (1atm)
Lindlar catalyst
N3
O
O
O
O
O
OH
OAc
O
O
O
Br
NaN3
DMF
Ac2O
HO
Br
EtOH
Py
DCM, BF3.Et2O
HO
AcO
AcO
AcO
AcO
OH
1
OAc
2
OAc
3
OAc
4
OAc
5
DMF
Folic acid, DCC
OH
OH
O
OAc
HO
HN
O
OH
O
OAc
OAc
H
N
H
N
OH
O
HN
O
O
NaOMe
MeOH
O
O
OH
O
OH
N
O
N
N
N
N
N
N
N
N
H
H
6
7
H2N
N
H2N
Figure 1. The synthesis route of FA-2-DG. Ac2O: acetic anhydride, Py: pyridine, DCM: dichloromethane,
DMF: dimethylformamide, DCC: dicyclohexylcarbodiimide.
2.2. Characterization of the Nano-Properties
To evaluate the self-assembly performance of FA-2-DG, we characterized its nano-properties.
Three different methods were applied: dynamic light scattering (DLS), transmission electron
microscopy (TEM) and atomic force microscopy (AFM).

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The DLS results showed that the peak diameter of FA-2-DG nano-particles in water was
approximately 78.8 nm, as shown in Figure 2A. Only one narrow peak in the graph was observed,
thus indicating that the particles were fairly homogeneous. The TEM results showed that the FA-2-DG
self-assembled into nano-fibers, as shown in Figure 2B. The nano-fibers further assembled into
nanoclusters. The positions shown by the blue arrow in Figure 2B indicate that the nano-fibers had
diameters ranging from 5 nm to 20 nm, indicating the assembly process of continuous aggregates from
fine to coarse. The AFM scans displayed the three-dimensional features of the sample, as shown in
Figure 2C. FA-2-DG also formed nano-fibers and at the ends of the fibers (the positions indicated by
the red arrows in Figure 2C), nano-particles with sizes of 20–30 nm were also found. The upper-left
part of Figure 2C shows that the depth of particles was around 30 nm, and the upper-right part of
the figure shows a particle diameter of around 25 nm. By plotting the absorbance of a set of FA-2-DG
solutions as a function of FA-2-DG concentration, the critical aggregation concentration of FA-2-DG
was 7.94 × 10−3 mM.
From all the three characterizations, the particle morphology and self-assembly capability of
FA-2-DG was confirmed, and the nano-scale assembly results were validated. The DLS results showed
the highest particle size, and the AFM results showed the largest particle size; the reason for this result
was the differences in sample states. The TEM and AFM results were consistent: both images showed
nano-fibers self-assembled from FA-2-DG.
Figure 2. Nano-properties of FA-2-DG, (A) DLS size analysis of FA-2-DG in water solution; (B) TEM
micrograph of FA-2-DG; (C) AFM image of FA-2-DG.
2.3. Cell Viability Assays
In vitro 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cytotoxicity
assays were used to evaluate the biological effects of FA-2-DG on cell viability. In addition to
a FA-2-DG and the blank control group, a third group treated with a physical mixture of folic
acid and 2-DG (FA-2-DG/PM) was also evaluated. The results in Figure 3 showed that, compared
with 2-DG, FA-2-DG/PM caused a modest decrease in cell viability at the high concentration of
400 µM, but FA-2-DG clearly inhibited cell viability in both MCF-7 and SKOV-3 cells. The inhibition
of FA-2-DG/PM may have resulted from the weak hydrogen bond between folic acid and 2-DG.
With increased time and concentration, the inhibition of FA-2-DG in both cell lines increased.
On one hand, the nanoparticles showed advantages in anti-tumor treatment, but on the other hand,
the results confirmed that the target of action was to interfere with glycolysis, and a long time was
necessary to show better results. For the two cell lines, FA-2-DG showed better (no statistical analysis)
inhibition than did SKOV-3 cells, because SKOV-3 cells expressed higher levels of folate receptors.
Therefore, the SKOV-3 cell line was used for further flow cytometric analysis. Because 2-DG did not
influence cell viability at the tested concentrations, it was unnecessary to analyze the effects on the cell
cycle and cell apoptosis.

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Figure 3. Cell viability assays of different treatments. (A) MCF-7 cells were treated with different
groups of compounds for 24 h and 48 h. (
compounds for 24 h and 48 h. Cell viability was determined by MTT assays. The results are expressed
as a mean percentage of controls standard deviation (SD) in three separate experiments. Significance
B) SKOV-3 cells were treated with different groups of
±
was calculated by ANOVA and Duncan’s test (* p < 0.05, ** p < 0.01).
2.4. Effects of FA-2-DG on the Cell Cycle in SKOV-3 Cells
To investigate whether FA-2-DG affected the cell cycle distribution of SKOV-3 cells, we treated
the cells with different groups of compounds for 24 h and then analyzed cell cycle progression using
flow cytometry. As shown in Figure 4A, FA-2-DG showed clear blocking of the cell cycle, and the
frequency of S-phase cells increased significantly. Figure 4B showed the proportion in each cell cycle
after treatment with three different groups. These results provide strong evidence of interference with
energy metabolism, ineffective DNA replication due to decreased ATP production, and more cells
arrested in S-phase.
Figure 4. The effect of FA-2-DG on cell cycle regulation in SKOV-3 cells. (A) The cells were treated
with the blank control and the other two groups of compounds at concentrations of 400
µM for 24 h,
stained with propidium iodide (PI) and then subjected to flow cytometry analysis to determine the
cell distribution at each phase of the cell cycle. The representative results from three independent
experiments are shown. (
Significant difference from the control was determined by using Student’s t-test (** p < 0.01).
B
) The results are expressed as means
±
SD of three independent experiments.

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2.5. Effects of FA-2-DG on the Induction of Apoptosis in SKOV-3 Cells
To further confirm that the FA-2-DG induced cell death was apoptosis, we performed flow
cytometry, using Annexin V and PI staining. As shown in Figure 5A, the ratio of late apoptotic cells
(upper right quadrant, Annexin V/PI positive) increased from 2.65% to 9.08% after 48 h of exposure
to 400 µM of FA-2-DG. Figure 5B showed the proportion of cells in different apoptotic stages after
three different treatments. Although the result of 9.08% did not provide clear evidence that FA-2-DG
induced apoptosis, compared with FA-2-DG/PM, the effect was statistically highly significant.
Figure 5. The effect of FA-2-DG on apoptosis in SKOV-3 cells. (
control and the other two groups of compounds at concentrations of 400
Annexin V and PI. ( ) The effect of FA-2-DG on cell death was determined via Annexin V-FITC/PI
analysis by using flow cytometry. The representative results from three independent experiments are
shown. ( ) The results are expressed as means SD of three independent experiments. Significant
difference from the control was determined by using Student’s t-test (** p < 0.01).
A
) The cells were treated with the blank
µM for 48 h, then stained with
A
B
±
2.6. Docking Studies
Docking studies were performed to verify that FA-2-DG targets folate receptors. As positive
controls, a folic acid and folate receptor (PDB ID:4LRH) docking model was used with preferred
docking orientation, as shown in Figure 6A; the cdocker energy was
interaction energy was 61.5 Kcal
mol−1. FA-2-DG docked into the interaction sphere in the same
manner as folic acid, as shown in Figure 6B; the cdocker energy was 56.5 Kcal
mol−1, and the
interaction energy was 59.5 Kcal
mol−1. To further understand the binding mode of FA-2-DG to the
−
59.5 Kcal
·
mol−1, and the
−
·
−
·
−
·
folate receptor, we performed a careful inspection of the docking pose in Figure 6C,D. In addition to
folic acid and receptor interactions, FA-2-DG showed more interactions with amino acids than folic
acid in the receptor, such as TYR60, SER101 and TRP134, suggesting that FA-2-DG can target the folate
receptor in a more firm way than folic acid.

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Figure 6. Docking studies of FA-2-DG with the folate receptor. (A) Folic acid docking into the folate
receptor, red color parts represents the alpha helix in the protein structure, light blue color parts
represents the beta folding; ( ) FA-2-DG docking into the folate receptor, red color parts represents
the alpha helix in the protein structure, light blue color parts represents the beta folding; ( ) the 2-D
) 2-D diagram of
B
C
diagram of ligand–receptor interactions between folic acid and the folate receptor; (
D
ligand–receptor interactions between FA-2-DG and the folate receptor.
3. Materials and Methods
3.1. Materials
Folic acid and 2-DG were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals,
solvents and biochemical reagents were of analytical grade and were purchased from commercial
sources (J&K Scientific, Beijing, China; Solarbio Science & Technology, Beijing, China).
3.2. Preparation of FA-2-DG
3.2.1. Preparation of (4R,5S,6R)-6-(acetoxymethyl) tetrahydro-2H-pyran-2,4,5-triyl triacetate
(compound 2)
A solution of 5.0 g (30.5 mmol) of 2-DG (compound 1) and 25 mL pyridine was prepared. To that
solution, 23.8 g (233 mmol) of acetic anhydride was added dropwise at 0 ◦C. The reaction mixture
was stirred at 25 ◦C for 16 h. After evaporation, the residue was diluted with 100 mL ethyl acetate.
The organic layers were washed with citric acid (30 mL
×
3), NaHCO3 (30 mL
×
3) and brine
(30 mL × 3), and then dried with anhydrous Na2SO4. After filtration, the filtrate was evaporated, and
the residue was purified by column chromatography (SiO2, petroleum ether/ethyl acetate = 10/1 to
5/1) to obtain acetyl 2-DG (compound
yield) as a colorless oil. H-NMR (400 MHz, CDCl3):
2
shown in synthetic route in Figure 1, 9.1 g, 27.39 mmol, 89.9%
/ppm = 5.77 (dd, J = 10.0, 2.20 Hz, 1H), 5.08–4.97
1
δ
(m, 2H), 4.31–4.26 (m, 1H), 4.01–4.03 (m, 1H), 3.74–3.70 (m, 1H), 2.35–2.29 (m, 1H), 2.09–1.90 (m, 12H).
13C-NMR (100 MHz, CDCl3):
δ/ppm = 170.71, 170.10, 169.76, 168.79, 91.08, 72.87, 70.15, 68.26, 61.95,
34.73, 20.96, 20.86, 20.77, 20.71. HRMS (m/z): calcd for C14H20O9 [M + Na]+: 355.0999, found: 355.1008.
3.2.2. Preparation of (2R,3S,4R)-2-(acetoxymethyl)-6-(2-bromoethoxy) Tetrahydro-2H-Pyran-3,4-diyl
Diacetate (Compound 3)
To a solution of compound
2
(5.0 g, 15 mmol), 2-bromoethanol (2.3 g, 18 mmol, 1.3 mL)
◦
in dichloromethane (35 mL) was added dropwise to BF3.Et2O (2.6 g, 18 mmol, 2.2 mL) at 0 C.
◦
After stirring at 25 C for 2 h, the reaction mixture was diluted with ice saturated NaHCO3 water
solution (60 mL) and extracted with dichloromethane (50 mL
×
2). The combined organic layers

Int. J. Mol. Sci. 2019, 20, 697
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were washed with brine (40 mL
×
3), then dried over anhydrous Na2SO4. After filtration, the
filtrate was evaporated, and the residue was purified by column chromatography (SiO2, petroleum
ether/ethyl acetate = 10/1 to 5/1) to obtain compound (4.6 g, 10.4 mmol, 69.3% yield, 90.0% purity) as
a yellow oil [26]. 1H-NMR (400 MHz, CDCl3):
/ppm = 5.33–5.26 (m, 1H), 5.01–4.95 (m, 2H), 4.28–4.24
(m, 1H), 4.08–4.04 (m, 2H), 3.94–3.88 (m, 1H), 3.82–3.76 (m, 1H),3.48 (t, J = 0.8 Hz, 2H), 2.29–2.25
(m, 1H), 2.11–2.98 (m, 9H), 1.85–1.76 (m, 1H). 13C-NMR (100 MHz, CDCl3):
/ppm = 170.71, 170.18,
3
δ
δ
169.93, 97.25, 69.24, 68.91, 68.31, 67.97, 62.33, 34.88, 30.09, 20.98, 20.78, 20.75. HRMS (m/z): calcd for
C14H21BrO8 [M + Na]+: 419.0312, found: 419.0324.
3.2.3. Preparation of (2R,3S,4R)-2-(acetoxymethyl)-6-(2-azidoethoxy) tetrahydro-2H-pyran-3,4-diyl
Diacetate (Compound 4)
Compound
3 (4.5 g, 11.3 mmol) and NaN3 (1.11 g, 16.9 mmol) in DMF (35 mL) were
◦
heated at 60 C for 16 h. The mixture was diluted with ice water (300 mL) and extracted with
ethyl acetate (100 mL
×
2). The combined organic layers were washed with brine (40 mL × 3)
,
dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to obtain
(2R,3S,4R)-2-(acetoxymethyl)-6-(2-azidoethoxy) tetrahydro-2H-pyran-3,4-diyl diacetate (3.9 g, crude)
1
as a yellow oil [27]. H-NMR (400 MHz, CDCl3):
δ/ppm = 5.23–5.21 (m, 1H), 4.92 (brs 2H), 4.23–4.19
(m, 1H), 3.99–3.92 (m, 2H), 3.77–3.73 (m, 1H), 3.60–3.51 (m, 1H), 3.36–3.35 (m, 2H), 2.23–2.19 (m, 1H),
2.01–2.91 (m, 9H), 1.79–1.72 (m, 1H). 13C-NMR (100 MHz, CDCl3):
/ppm = 170.71, 170.12, 169.93, 97.27,
δ
69.22, 68.83, 68.18, 66.55, 62.35, 50.44, 34.82, 20.96, 20.77, 20.74. HRMS (m/z): calcd for C14H21N3O8
[M + Na]+: 382.1221, found: 382.1211.
3.2.4. Preparation of (2R,3S,4R)-2-(acetoxymethyl)-6-(2-aminoethoxy) tetrahydro-2H-pyran-3,4-diyl
Diacetate (Compound 5)
Lindlar catalyst (0.6 g, 0.3 mmol, 10.0% purity) and p-TSA (0.19 g, 1.1 mmol) were added to
a solution of compound
4 (3.0 g, 9.2 mmol) in EtOH (70 mL). The mixture was stirred under H2
◦
(15 psi) at 25 C for 16 h. The mixture was diluted with ethanol and then filtered, and the filtrate
was concentrated under reduced pressure to obtain (2R,3S,4R)-2-(acetoxymethyl)-6-(2-aminoethoxy)
1
tetrahydro-2H-pyran-3,4-diyl diacetate (3.82 g, crude) as an off-white foam [28]. H-NMR (400 MHz,
CDCl3): δ/ppm = 7.71 (d, J = 8.0 Hz, 1H), 7.11 (d, J = 8.0 Hz, 1H), 6.38 (brs, 2H), 5.28–5.19 (m, 1H),
4.95–4.86 (m, 2H), 4.22 (m, 1H), 3.96–3.89 (m, 1H), 3.77–3.66 (m, 2H), 3.53–3.44 (m, 1H), 3.12–3.01
(m, 2H), 2.34 (s, 3H), 2.21 (m, 1H), 2.10–1.92 (m, 9H), 1.69–1.62 (m, 1H). 13C-NMR (100 MHz, CDCl3):
δ
/ppm = 170.77, 170.29, 169.81, 97.39, 69.07, 68.83, 68.18, 66.55, 62.35, 50.44, 34.82, 20.96, 20.77, 20.74.
HRMS (m/z): calcd for C14H24NO8 [M + H]+: 334.1496, found: 334.1506.
3.2.5. Preparation of N2-(4-(((2-amino-4-hydroxypteridin-6-yl) methyl) Amino)
benzoyl)-N5-(2-(((2S,4R,5S,6R)-4,5-diacetoxy-6-(acetoxymethyl) tetrahydro-2H-pyran-2-yl) oxy)
ethyl)-L-glutamine (Compound 6)
Compound
5 (1.75 g, 3.5 mmol) and DCC (1.5 g, 7.2 mmol) were added to a solution of folic acid
(1.25 g, 2.88 mmol) in DMSO (100 mL) and pyridine (20 g, 0.25 mol, 21 mL). The mixture was stirred
at 25 ◦C for 16 h in darkness. Then the reaction mixture was concentrated under reduced pressure
to remove most of the pyridine. The residue was washed with MTBE (500 mL) to obtain the crude
1
product compound
6
(4.4 g, crude) as a red solid. H-NMR (400 MHz, DMSO):
δ
/ppm = 11.44 (s, 1H),
8.64 (s, 1H), 7.95 (m, 1H), 7.66 (d, J = 8 Hz,1H), 7.19 (s, 1H), 7.13 (s, 1H), 7.07 (s, 1H), 6.93(s, 1H), 6.63
(t, J = 8 Hz, 1H), 5.14 (m, 1H), 4.93 (s, 1H), 4.82 (m, 1H), 4.48 (s, 1H), 4.35 (m, 1H), 4.16 (m, 1H), 3.97–3.91
(m, 2H), 3.56 (m, 1H), 3.42 (m, 1H), 3.28 (m, 1H), 2.54 (s, 1H), 2.26(m, 1H), 2.08 (m, 1H), 2.01 (s, 6H),
1.98 (s, 3H). 13C-NMR (100 MHz, DMSO):
δ/ppm = 174.52, 172.38, 170.58, 170.15, 169.87, 166.68, 158.13,
151.23, 148.99, 129.51, 128.41, 121.82, 118.56, 117.06, 112.86, 111.59, 96.66, 69.43, 68.98, 67.76, 66.27,
63.89, 62.48, 53.23, 46.38, 42.58, 38.93, 34.82, 30.98, 27.45, 21.12, 20.97, 20.91. HRMS (m/z): calcd for
C33H40N8O13 [M + H]+: 757.2788, found:757.2799.

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3.2.6. Preparation of N2-(4-(((2-amino-4-hydroxypteridin-6-yl) methyl) amino)
benzoyl)-N5-(2-(((2S,4R,5S,6R)-4,5-dihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl) oxy)
ethyl)-L-glutamine (Target Compound)
To a solution of compound
6
(3 g, 4 mmol) in MeOH◦(61 mL), NaOMe (6.23 g, 28.8 mmol, 25.0%
purity of MeOH) was added dropwise and stirred at 0 C. The reaction mixture was then stirred
at 25 ◦C for 1 h, acidified with acidic resin until pH = 6 was reached, and filtered. The filtrate was
concentrated under reduced pressure to yield a crude product. The crude product was purified with
a prep-HPLC (column: Agela Durashell (Bonna-Agela, Tianjin, China) 10
µ
m, 250
×
50 mm; mobile
phase: [water (10 mM NH4HCO3) -ACN]; B %: 0–15.0%, 20 min). After prep-HPLC purification,
the eluent was concentrated or evaporated to remove organic solvents. The residual aqueous solution
was lyophilized to obtain the product. 1H-NMR (400 MHz, CDCl3):
δ/ppm = 8.97 (s, 1H), 8.58
(d, J = 20 Hz, 2H), 7.95 (d, J = 10.6 Hz, 1H), 7.85 (s, 1H), 7.68–7.46 (m, 4H), 6.90 (s, 1H), 6.78 (s, 1H),
6.61 (dd, J = 19.0, 8.0 Hz, 2H), 4.78 (s, 1H), 4.46 (s, 1H), 4.34 (s, 1H), 4.20 (s, 1H), 4.06 (s, 1H), 3.64–3.21
(m, 11H), 3.02–2.98 (m,1H), 2.19–2.11 (m, 2H), 1.98 (s, 1H), 1.39 (t, J = 12.4 Hz, 1H). 13C-NMR (100 MHz,
DMSO): δ/ppm = 172.81, 166.64, 162.19, 156.88, 155.29, 151.01, 148.83, 129.48, 128.91, 128.41, 121.99,
111.81, 97.19, 73.62, 72.21, 68.38, 65.73, 65.52, 61.47, 54.43, 46.43, 38.91, 38.31, 33.01, 32.69, 29.08, 27.73.
HRMS (m/z): calcd for C27H34N8O10 [M + H]+: 631.2471, found: 631.2473.
3.3. Characterization of Nano-Properties
The nano-properties of FA-2-DG were evaluated by TEM, AFM imaging and DLS measurements.
3.3.1. Transmission Electron Microscopy
The TEM images were recorded on a JEM 2010 microscope (Jeol Ltd., Tokyo, Japan). Briefly, a drop
of the sample was placed on a copper grid coated with a carbon film and was allowed to dry for 5 min.
In a random region, the features and sizes of the nano-particles were identified by examination of
>100 species. An imaging plate with an energy window of 20 eV (Bioscan Camera Model 792; Gatan,
Pleasanton, CA, USA) was used to record 6000–40,000× digitally enlarged images [29].
3.3.2. Atomic Force Microscopy
The AFM measurements were recorded on a Multimode 8 instrument (BRUKER, Santa Barbara,
CA, USA) in contact mode, and the sample was prepared as a drop of the solution of FA-2-DG at
a concentration of 1.6 × 10−2 µM placed on a smooth mica plate [30].
3.3.3. Particle Size
A Zetasizer Nano ZS (Malvern, UK) instrument was used to measure the particle size of FA-2-DG
at the concentration of 1.6 × 10−3 mM [31].
3.4. Biological in Vitro Assays
3.4.1. Cell Culture
The MCF-7 cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM, Gibco; Thermo
Fisher Scientific, Inc.) supplemented with 100
µg/mL streptomycin, penicillin (Sigma-Aldrich;
Merck KGaA, Darmstadt, Germany) and 10% (v/v) FBS (Gibco; Thermo Fisher Scientific, Inc.). The cells
were cultured at 37 ◦C and 5% CO2 in a humidified environment [32].
The SKOV-3 cells were cultured in minimum essential medium (MEM) containing 5% fetal calf
serum, L-glutamine (2 mM) and a combination of streptomycin 0.1 mg/mL and penicillin 100 U/mL.
The cells were incubated at 37 ◦C in a humidified air atmosphere containing 5% CO2 [33].

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3.4.2. Cell Viability Assays
The cell viability of the compounds to the MCF-7 (human breast cancer) and SKOV-3
(human ovarian cancer) cells was determined by MTT assays. Cisplatin was a positive control and
blank DMSO was a negative control. Briefly, the cells were cultured in 96-well plates at a suitable
density (3000 cells per well) for 24 h before treatment. The solutions for each treatment group were
then added. After incubation for the given period, the culture medium was replaced by 100
MTT at 0.5 mg/mL, and the cells were incubated for a further 4 h. The medium solution was removed,
and an aliquot of 100 L DMSO was added. The cytotoxicity against the tested cells was measured
µL of
µ
at 570 nm with an auto-microplate reader (Infinite M200, Tecan, Männedorf, Switzerland) after the
crystals in the wells were dissolved by gentle shaking [34].
3.4.3. Flow Cytometric Analysis of the Cell Cycle and Measurement of the Apoptotic Ratio
Tumor cells were plated in six-well plates at 3
×
105 cells per well. After 24 h incubation, cells
in each group were treated for 24 h to analyze the cell cycle and for 48 h to measure cell apoptosis.
Cells incubated in medium without treatment were used as controls. For cell cycle analysis [3◦5],
the cells were harvested with trypsin, washed with cold PBS and fixed with 70% ethanol at 4 C
overnight. Before analysis, the cells were washed with PBS and stained with 50
µg/mL PI and
50
µ
g/mL RNase at 37 ◦C for 15 min. Finally, samples were immediately measured with a CytoFLEX
cytometer (Beckman Coulter, Pasadena, CA, USA). For cell apoptotic measurements [36], the cells
in different treated groups were harvested, washed with PBS and then treated with trypsin-EDTA
solution to detach the cells. The suspended cells were centrifuged at 200
washed twice with cold PBS, cells were collected by centrifugation at 200
FITC-conjugated Annexin V and PI. Then, the samples were immediately measured with a CytoFLEX
cytometer (Beckman Coulter, Pasadena CA, USA).
×
g for 10 min. After being
g for 5 min and stained with
×
3.5. Docking Simulation of FA-2-DG and Folate Receptor
The simulation of FA-2-DG docking onto the folate receptor was performed in Discovery Studio 4.5
(San Diego, CA, USA). The 3-D structure of the folate receptor was from the crystal structure of human
FRα in complex with folic acid (PDB ID: 4LRH), as a docking pocket, and the binding site of folic
acid to the folate receptor was predefined [37]. To prepare FA-2-DG for the docking simulation,
we first converted the 2-D structure to a 3-D structure, and energy minimization was performed.
The prediction of FA-2-DG docking onto the folate receptor was analyzed with CDOCKER on the basis
of the CHARMM force field [38,39].
3.6. Statistical Analysis
All results are shown as means and standard deviations calculated from the measurements, and
all experiments were carried out in three replicates to obtain these measurements. Data were analyzed
via Student’s t-test (comparing two samples) or ANOVA (comparing more than two samples), with
multiple pairwise comparisons being carried out using Duncan’s multiple range test. The means were
considered significantly different if * p < 0.05, ** p < 0.01 or *** p < 0.001.
4. Conclusions
In this work, folic acid-modified 2-DG (FA-2-DG) was synthesized, and its targeting ability
was evaluated through a computational study. FA-2-DG exhibited self-assembly ability and formed
nano-particles in water. It inhibited cell viability by disturbing glycolysis and blocking the cell cycle,
and it induced cell apoptosis. A computational study showed that FA-2-DG can dock onto the folate
receptor in the same manner as folic acid. All the results obtained from this study confirmed that
FA-2-DG, as a nano-scale pharmacosome, is a candidate for combination targeting anti-tumor therapy.

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Author Contributions: S.J. and P.W. conducted the conceptualization. Z.D. and S.J. completed the synthesis,
characterization and evaluation. H.G. validated the results. H.Z. and S.J. conducted the computation. S.J. and F.R.
wrote and prepared the original draft. P.W. reviewed and edited the manuscript. P.W. supervised the project. P.W.
took part in project administration and funding acquisition.
Funding: This study was supported by the 111 project from the Education Ministry of China (No. B18053).
Conflicts of Interest: The authors declare no conflict of interest.
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