d-Fructose Modification Enhanced Internalization of Mixed Micelles in Breast Cancer Cells via GLUT5 Transporters

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Bioscience

Full Paper

d-Fructose Modiﬁcation Enhanced Internalization

of Mixed Micelles in Breast Cancer Cells via

GLUT5 Transporters

Xu Zhou, Xianyan Qin, Tao Gong, Zhi-Rong Zhang, Yao Fu*

d-Fructose modiﬁed poly(ε-caprolactone)-polyethylene glycol (PCL-PEG-Fru) diblock amphi-

phile is synthesized via Cu(I)-catalyzed click chemistry, which self-assembles with D-α-

tocopheryl polyethylene glycol 1000 succinate (TPGS) into PCL-PEG-Fru/TPGS mixed micelles

(PPF MM). It has been proven that glucose transporter (GLUT)5 is overexpressed in MCF-7 cells

other than L929 cells. In this study, PPF MM exhibit a signiﬁ-

cantly higher uptake efﬁciency than fructose-free PCL-PEG-

N₃/TPGS mixed micelles in both 2D MCF-7 cells and 3D tumor

spheroids. Also, the presence of free ^d-fructose competitively

inhibits the internalization of PPF MM in MCF-7 cells other

than L929 cells. PPF MM show selective tumor accumula-

tion in MCF-7 breast tumor bearing mice xenografts. Taken

together, PPF MM represent a promising nanoscale carrier

system to achieve GLUT5-mediated cell speciﬁc delivery in

cancer therapy.

1. Introduction

cells, resulting from overexpressed GLUT1, GLUT2, and/

or GLUT5 in different tumor cells.^[6,7]As a result, such dis-

criminators of tumors from normal tissues can be explored

as potential targets for developing tumor-speciﬁc delivery

strategies.

As a vital source of energy for life, carbohydrates are crit-

ical to maintain the normal structure and function of cells,

tissues, and organs. Cells take up monosaccharaides in a

concentration-dependent manner via glucose transporters

(GLUTs).^[1,2]Among 14 GLUT transporters identiﬁed, each

shows different tissue distribution pattern, gene regula-

tion, substrate preference, and kinetic properties.^[3,4]Often,

the altered activity of the GLUT transporters is correlated

with various diseases and disorders. In 1920s, Otto War-

burg and colleagues found that under aerobic conditions,

tumors consume more glucose for energy production than

normal tissues.^[5]Accelerated metabolism and increased

glucose uptake are typical among most malignant tumor

Over the years, breast cancer has been one of the

most commonly diagnosed malignancy among women

with increasing incidence globally.^[8]Studies found that

GLUT1, GLUT2, and GLUT5 overexpress in breast cancer

cells, while GLUT5 is absent in normal breast tissues.^[2,9,10]

Hence, GLUT5, a fructose-speciﬁc transporter, has become

a target of extensive research interests due to its overex-

pression in breast cancer cells. To use fructose as the spe-

ciﬁc ligand for GLUT5, Stenzel and co-workers synthesized

various poly (1-O-methacryloyl-β-d-fructopyranose)-b-

poly-(methyl methacrylate) copolymers to assemble into

spherical micelles which showed speciﬁc cell uptake in

breast cancer cell lines including MCF-7 and MDA-MB-

231 but not in macrophage RAW264.7.^[10]Next, Zhao

et al. fabricated rod-shaped fructose micelles of varying

structures and size distributions which showed length-

dependent cellular uptake and movement behavior in

X. Zhou, X. Qin, Prof. T. Gong, Prof. Z.-R. Zhang, Dr. Y. Fu

Key Laboratory of Drug Targeting and Delivery Systems

West China School of Pharmacy

Sichuan University

Chengdu 610041, China

E-mail: yfu4@scu.edu.cn

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vitro.^[11]Also, ﬂuorinated fructose analogues have been

developed for positron emission tomography-imaging in

cancer diagnosis of GLUT5-overexpressing tumors.^[12]Car-

boxyl end-functionalized poly(1-O-methacryloyl-2,3:4,5-

di-O-isopropylidene-β-d-fructopyranose) was prepared

and grafted onto the surface of amine-functionalized

nanodiamonds to be applied to human breast cancer

therapy.^[13]These studies strongly support that the pres-

ence of fructose in the polymer or small molecule conju-

gate is critical to achieve GLUT5-mediated cellular uptake

in GLUT5-overexpressing tumor cells.

Polymeric micelles consisting of amphiphiles are typi-

cally 20–100 nm in diameter.^[14–18]These self-assembled

nanostructures have hydrophilic surfaces exhibiting

prolonged circulation in vivo.^[19]By conjugating the tar-

geting ligand to the hydrophilic head of the polymer,

ligands will be exposed outward after self-assembly; thus

rendering micelles more likely to interact with recep-

tors of interests. Cao et al. previously reported a D-α-

tocopheryl polyethylene glycol 1000 succinate (TPGS)/

pluronic mixed micelle system with highly efﬁcient cell

uptake in 2D and outstanding tumor penetration in 3D

conditions.^[15]Combining two or more clinically approved

materials, mixed micelles thus present a versatile and

ﬂexible delivery platform with tunable size, morphology,

and functionality.

Polycaprolactone (PCL) is a biodegradable polymer that

has been approved by the US Food and Drug Administra-

tion for applications in the human body such as sutures,

tissue scaffolds, and adhesion barriers.^[20–22]PCL-derived

diblock copolymers have been extensively used in con-

trolled release and targeted drug delivery.^[23–26]Herein,

we report the synthesis of a ^d-fructose conjugated diblock

copolymer poly(ε-caprolactone)-poly(ethylene glycol)

(PCL-PEG-Fru) via click chemistry. Next, PCL-PEG-Fru/TPGS

mixed micelles were fabricated to achieve GLUT5-medi-

ated cell speciﬁc uptake in breast cancer cells. Together,

our study demonstrates that PCL-PEG-Fru/TPGS mixed

micelles greatly improve the afﬁnity to breast cancer cells

thus representing a viable strategy to achieve targeted

therapy of breast cancer.

Cell Signaling Technology (Danvers, MA, USA). 4′,6-Diamidino-

2-phenylindole dihydrochloride (DAPI) was purchased from

Beyotime (Haimen, China). Benzyl alcohol was of analytic grade

which was distilled before use. All other chemicals and solvents

were of analytical grade.

NMR spectrometer (Agilent DD2-600/54, Agilent Technologies,

USA); mass spectrometer (Agilent 6410B, Agilent Technologies,

USA); gel permeation chromatography (Waters 515 HPLC Pump,

Waters 2410 Refractive Index Detector, Waters, USA); dynamic

light scattering (DLS) (Malvern Zetasizer Nano ZS, Malvern, UK);

transmission electron microscope (TEM) (H-600, Hitachi, Japan);

ﬂuorescence spectrophotometer (RF-5301PC, Shimadzu, Japan);

confocal laser scanning microscopy (CLSM) (FV1000, Olympus,

Japan); ﬂow cytometry (Beckman Coulter, Fullerton, CA, USA);

and IVIS Lumina Series III (Perkin Elmer, USA).

2.2. Synthesis of PCL-PEG-N₃and PCL-PEG-Fru

The synthetic routes of 3-O-propinyl-β-d-fructopyranose

and PCL-PEG-Fru are outlined in Scheme 1. 3-O-propinyl-β-d-

fructopyranose was synthesized according to previously reported

procedures.^[27]

2.2.1. Synthesis of 1,2:4,5-di-O-isopropylidene-β-d-

fructopyranose (1)

d-Fructose (10 g, 0.055 mol) was suspended in acetone (200 mL),

and stirred at room temperature. 14 mL of concentrated sulfuric

acid was added to the mixture until the fructose was dissolved

completely. 125 mL of sodium hydroxide solution (4.5 mol L⁻¹

)

was added dropwise to the reaction mixture at 0 °C. The mix-

ture was then concentrated under vacuum at 50 °C and extracted

with dichloromethane three times. The organic phase was com-

bined and washed with brine, dried over anhydrous sodium

sulfate, ﬁltrated, and concentrated under reduced pressure. The

crude product was recrystallized out of hexane:diethyl ether

(1:1, v/v) to yield compound 1 (9.8 g, 67.8%). The structure of

compound 1 was veriﬁed using a NMR spectrometer. ¹H-NMR

(400 MHz, CDCl₃): δ4.57–4.63 (dd, 1H); δ4.22–4.36 (m, 2H); δ3.91–

3.93 (dd, 1H); δ3.76–3.78 (m, 1H); δ3.68–3.71(dd, 2H); δ1.89–1.92

(s, 1H); δ1.40–1.48 (m, 12H). ¹³C NMR (151 MHz, CDCl₃): δ (ppm)

109.17, 108.63, 103.17, 71.08, 70.89, 70.14, 65.61, 61.37, 26.57,

25.87, 25.45, and 24.07. The mass and molecular formula were

determined by a mass spectrometer. m/z 283[M+23]⁺: calcd. for

C₁₂H₂₀O₆, 260; observed, 260 (Figure S5, Supporting Information).

2. Experimental Section

2.2.2. Synthesis of 3-O-propinyl-1,2:4,5-di-O-

isopropylidene-β-d-fructopyranose (2)

2.1. Materials and Analytical Instruments

1,2:4,5-di-O-isopropylidene-β-d-fructopyranose (4 g, 0.015 mol)

was dissolved in 40 mL of dimethylformamide (DMF), added with

sodium hydroxide solid (1.74 g, 0.0435 mol). After stirring on ice

bath for 15 min, propargyl bromide (2 mL) was added slowly.

After stirring at room temperature for 24 h, 150 mL of ultrapure

water was added to the reaction solution. The mixture was

extracted with diethyl ether three times, the organic phase was

then washed with 10% hydrochloric acid three times, and satu-

rated salt water dried over anhydrous sodium sulfate, ﬁltrated,

Bifunctional NH₂-PEG₅₀₀₀-N₃(M_w, 5000 Da) was obtained from

Jenkem Technology (Beijing, China). ε-Caprolactone, stannous

(II) octoate (SnOct), TPGS, coumarin-6 (C6), and 1,1-dioctadecyl-

3,3,3′,3′-tetramethyl indodicarbocyanine (Did) were purchased

from Sigma-Aldrich (St Louis, MO, USA). Paclitaxel was provided

by Xi’an Haoxuan Biotechnology (Xi’an, China). GLUT5 antibody

was obtained from Santa Cruz Biotechnology (USA). Anti-rabbit

IgG Fab2 Alexa Fluor 647 molecular probes were obtained from

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Scheme 1. (1) Synthesis of 3-O-propinyl-β-d-fructopyranose. Reagents and conditions: (a) acetone, NaOH, H₂SO₄; (b) bromine propiolic, NaOH,

dimethylformamide; (c) triﬂuoroacetic acid, H₂O. (2) Synthesis of PCL-PEG-Fru. Reagents and conditions: (a) 130 °C, 24 h; (b) pyridine, dichlo-

romethane, 24 h; (c) triethylamine, dichloromethane, 24 h; (d) PMDETA, CuBr, dimethylformamide, 48 h.

and concentrated under reduced pressure to yield compound 2

(3.4 g, 74.2%).^[10]The structure of compound 2 was veriﬁed using

¹H-NMR (400 MHz, CDCl₃): δ4.57–4.63 (m, 1H); δ4.32–4.36 (m,

1H); δ4.24–4.28 (m, 2H); δ3.86–4.02 (t, 2H); δ3.70–3.74 (m, 2H);

δ3.65–3.69 (s, 1H); δ2.41–2.45 (t, 1H); δ1.3–1.58 (m, 12H). ¹³C NMR

(151 MHz, CDCl₃): δ (ppm) 109.03, 108.66, 102.61, 79.45, 74.68,

71.19, 71.03, 70.33, 70.25, 61.10, 59.12, 26.64, 25.96, 25.32, and

24.14. The mass and molecular formula were determined as m/z

321[M+23]⁺: calcd. for C₁₅H₂₂O₆, 298; observed, 298 (Figure S8,

Supporting Information).

(ii) the activation of PCL-OH with 4-nitrophenyl chloroformate

(NPC); (iii) the coupling of NPC-activated PCL with N₃-PEG₅₀₀₀

-

NH₂; (iv) the conjugation of 3-O-propinyl-β-d-fructopyranose

with PCL-PEG-N₃via Cu(I)-catalyzed click reaction.

Synthesis of PCL-OH: Monohydroxy-terminated PCL-OH

was ﬁrst synthesized by ring-opening polymerization of

ε-caprolactone using benzyl alcohol as an initiator and SnOct as a

catalyst (0.1% moles of ε-caprolactone) according to the literature.

In brief, ε-caprolactone (150 mmol, 17.27 g) and benzyl alcohol

(10 mmol, 1.08 g) were placed in a dry round-bottom ﬂask, and

0.1% amount of SnOct was added. Polymerization was carried out

under a dry nitrogen atmosphere and stirred constantly at 120 °C

for 24 h. The product was then dissolved in CH₂Cl₂and precipi-

tated three times in cold methanol. Finally, the white powder

was dried in a vacuum oven for 48 h at 40 °C. The structure of

PCL-OH was veriﬁed using ¹H NMR (400 MHz, CDCl₃, ppm): δ7.35

2.2.3. Synthesis of 3-O-propinyl-β-d-fructopyranose (3)

3-O-propinyl-1,2:4,5-di-O-isopropylidene-β-d-fructopyranose

(1 g, 0.0034 mol) was dissolved in triﬂuoroacetic acid (TFA):H₂O

(9:1, v/v). After stirring at room temperature for 1 h, the reac-

tion solution was ﬁltered and concentrated under reduced pres-

sure at 65 °C. The product was further puriﬁed by ﬂash column

chromatography with dichloromethane:methanol (20:1, v/v) as

eluent (yield 68.5%). The structure of compound 3 was veriﬁed



(t, 5H, C₆H₅), 5.12 (t, 2H, C₆H₅CH₂), 4.06 (t, 2H, CH₂O ), 2.32



(t, 2H, CH₂CO ), 1.64 (4H, m, CH₂), 1.38 (2H, m, CH₂). The

Mw of the PCL-OH synthesized was 4385, which was calculated



via the integration ratio of peaks at 5.12 ( CH₂C₆H₅) with those

1

using H-NMR (400 MHz, CDCl₃): δ4.28–4.32 (m, 2H); δ3.98–4.03



at 4.06 ( CH₂O ).

(m, 2H); δ3.86–3.89 (m, 2H); δ3.78–3.82 (s, 1H); δ3.65–3.69 (m,

1H); δ3.49–3.53 (m, 1H); δ2.47–2.50 (t, 1H); δ2.11–2.15 (t, 4H).

¹³C NMR (151 MHz, (CD₃)₂SO): δ (ppm) 98.01, 77.04, 76.20, 75.02,

73.85, 71.10, 67.62, 63.01, and 58.04. The mass and molecular

formula were determined as m/z 241[M+23]⁺: calcd. for C₉H₁₄O₆,

218; observed, 218 (Figure S11, Supporting Information).

Synthesis of Activated PCL: Under a nitrogen atmosphere, to a

10 mL CH₂Cl₂solution of PCL-OH (1 mmol) and pyridine (0.4 mL,

5 mmol), a solution of NPC (2.4 g, 6 mmol) in 10 mL of CH₂Cl₂

was added at 0 °C. Afterward, the reaction proceeded for 24 h at

room temperature. The resulting polymer PCL-NPC was isolated

by precipitation in cold diethyl ether and dried under vacuum.

1

The structure of PCL-NPC was veriﬁed using H NMR (400 MHz,



CDCl₃, ppm): 8.27–8.30 (m, 4H, C₆H₄NO₂), 7.38 (m, 5H, C₆H₅),

2.2.4. Synthesis of Poly(ε-caprolactone) Polyethylene

Glycol-Fructose



5.12 (t, 2H, C₆H₅CH₂), 4.07 (t, 2H, CH₂COOC₆H₄NO₂), 4.04 (t, 2H,



CH₂OH), 2.32 (t, 2H, CH₂CO), 1.64 (m, 4H, CH₂), 1.38 (m,



2H, CH₂). The new peak at δ8.27–8.30 was attributed to the

benzene ring of NPC, conﬁrming that PCL-NPC was successfully

synthesized.

The synthetic scheme for PCL-PEG-Fru is shown in Scheme 1.

Based upon a previously described method,^[26]the synthesis

of PCL-PEG-Fru includes four steps: (i) the synthesis of PCL-OH;

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Synthesis of PCL-PEG-N₃: The puriﬁed PCL-NPC (0.1 g) was mixed

with N₃-PEG₅₀₀₀-NH₂(0.15 g) in CH₂Cl₂and 250 µL of trimethylamine

was added. Afterward, the reaction was stirred for another 24 h

at room temperature. Then the reaction solution was transferred into

a dialysis tube (Molecular Weight Cut Off (MWCO) 8000–14 000 Da),

and dialyzed against double distilled water for 3 d. PCL-PEG-N₃was

collected as a white solid after freeze-drying (yield: 50%).

Brieﬂy, accurately weighed polymers and ﬂuorescent dyes were

dissolved in acetonitrile, and evaporated under vacuum at 50 °C.

The resultant thin ﬁlm was then hydrated with 2 mL of ddH₂O at

50 °C for 5 min to obtain a micelle suspension, which was then

ﬁltered through 0.22 µm ﬁlter membrane. As for the quantitative

analysis of the encapsulated probes, we used ethanol demulsiﬁ-

cation method to disrupt the micelles to determine the concen-

tration using ﬂuorescence spectrophotometer. The encapsulation

efﬁciencies of PPN MM and PPF MM were determined to be above

80% (Table S2, Supporting Information). The release proﬁles

of C6 from PPN MM and PPF MM were studied using a dialysis

setup. Speciﬁcally, 1 mL of micelle formulations were transferred

to dialysis tubings (MWCO 7 kDa), and subjected to dialysis

against 40 mL of phosphate buffer saline (PBS) containing 0.2%

Tween 80 (v/v). The sink condition was maintained under gentle

shaking at 100 rpm and 37 °C. The release media (1.0 mL) was

collected at predetermined time intervals and replaced with

equal volume of fresh medium. The concentrations of C6 at each

time point were determined as previously described, the cumula-

tive drug release percentage was calculated accordingly.^[29]

Conjugation of Compound 3 and PCL-PEG-N₃: 3-O-propinyl-

β-d-fructopyranose (436 mg, 2 mmol) and PCL-PEG-N₃(500 mg,

0.1 mmol) were dissolved in 20 mL of DMF, CuBr (288 mg, 2 mmol)

and 500 µL of N,N,N′,N″,N″-pentamethyldiethylenetriamine

(PMDETA) were then added. After the mixture was stirred for 48 h

at 60 °C, 40 mL of aqueous ammonia was poured into the reac-

tion solution and stirred for 20 min at room temperature. Then

the reaction solution was transferred into a dialysis tube (MWCO

8000–14 000 Da), and dialyzed against 0.5% ethylenediamine-

tetraacetic acid (EDTA) for 24 h and water for another 2 d. PCL-

PEG-Fru was collected as a white solid after freeze-drying (yield: 50%).

2.3. Preparation and Characterization of PCL-PEG/TPGS

Mixed Micelles

2.6. Cell Culture

PCL-PEG-Fru/TPGS mixed micelles (PPF MM) and PCL-PEG-N₃/TPGS

mixed micelles (PPN MM) were prepared by thin-ﬁlm hydration

method as previously described.^[15]Brieﬂy, accurately weighed

PCL-PEG-Fru and TPGS (10:1, w/w) were dissolved in acetonitrile,

and evaporated under vacuum at 50 °C. The resultant thin ﬁlm

was then hydrated with 2 mL of ddH₂O at 50 °C for 5 min to obtain

a micelle suspension, which was then ﬁltered through 0.22 µm

ﬁlter membrane. PPN MM was prepared similarly by using PCL-

PEG-N₃instead of PCL-PEG-Fru. The polymer concentrations of the

Human breast cancer cell line MCF-7, murine ﬁbroblast L929,

murine macrophage RAW264.7, and human umbilical vein

endothelial cells (HUVECs) were obtained from the Cell Bank

of Chinese Academy of Sciences (Shanghai, China). Cells were

cultured in RPMI-1640 medium (Gibco, USA) containing 10%

fetal bovine serum (Gibco, USA), 100 µg mL⁻¹streptomycin, and

100 U mL⁻¹penicillin. Cells were maintained at 37 °C in a humid-

iﬁed atmosphere containing 5% CO₂.

two mixed micelles were kept consistent at 5 mg mL⁻¹

.

The size and morphology of above mixed micelles were

characterized by DLS analysis, and TEM. DLS measurements were

performed in triplicates at 25 °C and 90° angle.

2.7. Immunoﬂuorescence Staining of GLUT5

To examine the expression of GLUT5 transporter on MCF-7 cells,

we used immunoﬂuorescence staining to label GLUT5 trans-

porter. L929 cells were used as control. Speciﬁcally, cells were

washed with PBS three times and ﬁxed in 4% paraformaldehyde

for 30 min, washed with PBS three times, treated with 0.3%

Triton-100 for 30 min, washed with PBS three times, and incu-

bated with 5% bovine serum albumin for 2 h. Then cells were

incubated with primary antibodies (dilution 1:100) overnight at

4 °C in a humidiﬁed chamber. Subsequently, cells were washed

with PBS three times and incubated with secondary antibodies

(Anti-rabbit IgG Fab2 Alexa Fluor 647 Molecular Probes) (dilu-

tion 1:250) for 60 min at room temperature. After staining with

20 µg mL⁻¹DAPI for 5 min, cells were observed under CLSM and

all parameters were maintained the same when taking images

including exposure time, HV, gain, and offset.

2.4. Determination of Critical Micelle Concentration (CMC)

The CMC was determined by a ﬂuorescence probe technique

using pyrene as a ﬂuorescence probe as previously described.^[28]

In brief, 100 µL of pyrene (≈3.0 µg mL⁻¹) in tetrahydrofuran (THF)

was added to each 5 mL volumetric ﬂasks and THF was subse-

quently removed by evaporation at room temperature in the

fume hood for 24 h. 5 mL of micelle samples (PPN MM and PPF

MM) of a series of concentrations was added to the above volu-

metric ﬂasks. The combined solutions of pyrene and micelles

were then sonicated for 30 min, and allowed to reach equilib-

rium by cooling overnight at room temperature before meas-

urement. CMC values were determined from the onset of a rise

in the intensity ratio of peaks at an emission wavelength of

338 nm (I₃) to peaks at excitation wavelength of 333 nm (I₁) in

the ﬂuorescence spectra of pyrene plotted versus the logarithm

of polymer concentrations.

2.8. Cellular Uptake, Intracellular Distribution, and

Internalization Pathway

MCF-7, RAW264.7, and L929 cells were seeded in 12-well plate

(5 × 10⁵cells per well), and exposed to C6-loaded PPN MM and

C6-loaded PPF MM with serum-free RPMI-1640. The same

concentrations of the micelles (ﬁnal concentration of C6 was

0.75 µg mL⁻¹) were added to the wells for given time periods

2.5. In Vitro Release

C6 was used as a ﬂuorescence probe in the cell based studies

to interpret the endocytosis behavior of PPN MM and PPF MM.

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such as 5 min, 1 h, 2 h, and 4 h. At given time points, the medium

was removed, and the cells were washed with ice-cold PBS three

times. Cells were then harvested by trypsin-EDTA treatment, and

cell pellets were collected by centrifugation followed by three

washes with ice-cold PBS. Finally, the intracellular ﬂuorescence

intensity of C6 in the sample was analyzed by ﬂow cytometry.

To evaluate whether free fructose can inhibit GLUT5-medi-

ated cell uptake, free d-fructose was preincubated with MCF-7

and L929 cells prior to exposure to C6-loaded PPN and PPF MMs.

Speciﬁcally, MCF-7 cells were preincubated with free d-fructose

diluted in serum-free RPMI-1640 for 1 h (ﬁnal concentrations of

d-fructose were 5.6 and 56 × 10⁻³m). Then cells were coincubated

with C6-loaded PPN MM and C6-loaded PPF MM (ﬁnal concentra-

tion of C6 was 0.50 µg mL⁻¹). After incubation for another 1 h at

37 °C, cells were rinsed, harvested, and collected by centrifuga-

tion. The ﬂuorescence intensity of C6 in the sample was analyzed

by ﬂow cytometry.

2.10. In Vivo Imaging of Micelles in MCF-7

Tumor-Bearing Nude Mice

Nude mice were purchased from Dashuo Biotechnology

(Chengdu, China) and maintained under standard housing con-

ditions with free access to food and water. All animal experi-

ments were approved by the Ethics Committee of Sichuan

University and conducted in accordance with institutional

guidelines.

A suspension of MCF-7 cells (0.1 mL, 5 × 10⁵cells) was injected

subcutaneously into the right ﬂank of nude mice to induce

growth of subcutaneous tumor xenografts. In vivo imaging and

biodistribution experiments were conducted approximately

four weeks after tumor cell inoculation, by which time tumors

reached ≈1 cm in diameter. A near infrared probe Did was encap-

sulated in the mixed micelles, which were then administered

to MCF-7 tumor-bearing nude mice via tail vein at a dose of

0.015 mg Did kg⁻¹. At 1, 4, 24, and 48 h after injection, mice were

narcotized for in vivo imaging (IVIS Lumina Series III). Then,

nude mice were sacriﬁced, and their tumors were collected for ex

vivo imaging.

To perform CLSM, at predetermined time points, cells were

rinsed with ice-cold PBS three times and ﬁxed with 4% paraform-

aldehyde for 30 min, stained with 20 µg mL⁻¹DAPI for 5 min, and

observed under CLSM.

To examine the internalization and subcellular localization

of PPN MM and PPF MM in MCF-7 cells, cell endosomes and lys-

osomes were labeled with LysoTracker red (Molecular Probes,

ThermoFisher, USA), and cotracked the green and red ﬂuores-

cence within cells. C6-loaded PPN MM and C6-loaded PPF MM

were administered at an equivalent concentration of 0.5 µg mL⁻¹

C6 for indicated time periods. After that, cells were rinsed three

times with ice-cold PBS and incubated with LysoTracker red for

30 min. The samples were then examined by CLSM.

2.11. Statistical Analysis

One-way ANOVA analysis was performed for comparison among

multiple groups followed by a Tukey post hoc analysis. All results

were presented as mean SD. A p-value of <0.05 was considered

statistically signiﬁcant.

For elucidating the endocytosis pathways, various

endocytosis inhibitors were selected including amiloride

(25 × 10⁻⁶m), chlorpromazine (20 × 10⁻⁶m), nystatin (40 × 10⁻⁶m),

M-β-CD (6.5 × 10⁻³m), and hypertonic sucrose (100 × 10⁻³m). MCF-7

cells were preincubated with each inhibitor for 1 h at 37 °C, fol-

lowed by incubation at 37 °C for another hour after addition of

C6-loaded PPN MM and PPF MM. After the harvest of cells, intra-

cellular C6 was determined by ﬂow cytometry.

3. Results and Discussion

3.1. Synthesis, Fabrication, and Characterization

of PCL-PEG/TPGS Mixed Micelles

PCL-PEG-N₃was synthesized by conjugating bifunctional

NH₂-PEG₅₀₀₀-N₃(M_w, 5000 Da) with 4-nitrophenyl chloro-

formate-activated PCL (PCL-NPC) (Scheme 1). The average

molecular weight of PCL-OH was about 4500 Da as deter-

mined by gel permeation chromatography (Table S1, Sup-

porting Information) and the average molecular weight of

each polymer was given in Figure S16 and Table S1 of the

Supporting Information. Next, PCL-PEG-Fru was synthe-

sized by conjugating PCL-PEG-N₃with 3-O-propinyl-β-d-

fructopyranose via click reaction.

2.9. Penetration of Micelles in Multicellular Tumor

Spheroids (MCTS)

To initiate the growth of MCTS, MCF-7 cells were seeded at a den-

sity of 2000 cells per well in 96-well plates precoated with 100 µL

of 2% low-melting-temperature agarose. Following cellular aggre-

gation and growth, each well contained a single spheroid, and

during growth 50% of the culture medium was changed with

fresh medium every other day. The experiment was started when

the tumor spheroids reached about 300–400 µm in diameter.

Next, MCTS were treated with free C6 solution, C6-loaded PPN

MM, and PPF MM at an equivalent concentration of 0.5 µg mL⁻¹

of C6 solution. After 5 min, 30 min, 4 h, and 24 h of incubation,

the spheroids were rinsed with ice-cold PBS for three times, and

then transferred to glass bottom petri-dishes, and covered with

glycerophosphate. Spheroid samples were then observed under

CLSM and the semi-quantitative analysis was carried out by

ImageJ, a professional software which can be used to quantify

ﬂuorescence intensity.

The mixed micelles were prepared using a thin-ﬁlm

hydration method. As shown in Figure 1, PPN MM dis-

played an average size distribution of 38.4 3.5 nm with

a polydispersity index (PDI) of 0.107, while PPF MM

showed an average size distribution of 57.1

3.0 nm

(PDI = 0.188). Regarding the TEM morphological character-

istics, both mixed micelles displayed near spherical shapes

and sizes, which were in accordance to the DLS meas-

urement (Figure 1A,B). The size distributions remained

unchanged over time, which indicates PPF MM and PPN

MM formulations are stable within the given period

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Figure 1. Characterizations of mixed micelles. A) Morphological images of PCL-PEG-N₃/TPGS mixed micelles (PPN MM) and PCL-PEG-Fru/

TPGS mixed micelles (PPF MM) by TEM. Scale bars represent 100 nm. B) Dynamic laser scattering (DLS) measurements of PPN MM and PPF

MM. C) Critical micelle concentrations of PPN MM and PPF MM. D) Size distributions of micelles at the given time by DLS.

(Figure 1D). The CMC values of two mixed micelles were

28.68 and 42.66 µg mL⁻¹, respectively, which were sufﬁ-

ciently low to maintain the micellar structure during the

following experiments. Moreover, Cao et al. previously

reported that the presence of TPGS could also improve the

stability of self-assemblies.^[15]

Next, we looked at the release of C6 from mixed

micelles. The cumulative release of C6 from the two

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micelle systems remained relatively

low (<18.0%) after 24 h (Figure S1, Sup-

porting Information), indicating that

the behavior of C6 could represent the

micelles in the cell study below.

At all given concentrations, the cell

viabilities of HUVECs were greater than

80% after 24 h, indicating that the blank

mixed micelles exhibited no obvious

cytotoxicity against HUVECs, L929,

RAW, and MCF-7 cells (Figure S2, Sup-

porting Information).

3.2. GLUT5 Transporters Overexpressed

in MCF-7 Cells

To conﬁrm whether MCF-7 cells can be

used as a model cell line to interpret

GLUT5-mediated cell uptake behavior,

we examined the level of GLUT5 expres-

sion on MCF-7 cells and L929 cells by

immunoﬂuorescence staining. L929

cells were used as a negative control

per previous report.^[9]Debating results

were observed and reported by different

groups. Vera and co-workers reported

Figure 2. Immunoﬂuorescence staining of GLUT5 in MCF-7 cells and L929 cells. Scale

bar represents 20 µm. The control section was incubated in the absence of the primary

antibody (2° Ab only).

the overexpression of GLUT5 in MCF-7

and MDA-468 breast carcinoma cell lines

in 1996.^[6]However, a study by Gambhir

and co-workers revealed that GLUT5 is not overexpressed

in breast cancer cells and patient breast cancer tissues.^[30]

In the present study, we investigated the level of GLUT5

expression in MCF-7 and L929 cells by immunoﬂuores-

cence staining. As shown in Figure 2, MCF-7 cells displayed

strong red ﬂuorescence compared to L929 cells, which

demonstrated a much higher level of GLUT5 expression

in MCF-7 cells. The overexpression indicated that GLUT5

could be a potential target site and PPF MM could achieve

GLUT5-mediated cellular uptake in MCF-7 cells.

Besides, the ﬂuorescence intensity of micelles taken up by

MCF-7 cells was much higher than those internalized by

RAW264.7 cells. The limited amount of the mixed micelles

taken up by RAW264.7 cells may suggest that the mixed

micelles were “invisible” to macrophages, which would

prolong their circulation in the blood system, and further

increase accumulation at the tumor site likely due to the

enhanced permeation and retention effect in vivo.

To explore whether GLUT5 is responsible for the

increased uptake efﬁciency of PPF MM as compared to

PPN MM, we carried out a competitive inhibition assay in

MCF-7 cells. Varying concentrations of ^d-fructose, i.e., 5.6

and 56 × 10⁻³m, were added to competitively inhibit the

internalization of PPF MM. Although the presence of free

D-frutcose did not entirely block the internalization of PPF

MM, the uptake of PPF MM distinctly decreased by 8.0%

and 26.0% in MCF-7 cells in a concentration-dependent

manner (Figure 4A). In comparison, the addition of free

d-fructose did not impact the uptake of PPN MM in MCF-7

cells (Figure 4A). In general, the cellular uptake could be

competitively inhibited by preincubation of free ^d-fruc-

tose indicating that PPF MM internalization was partially

mediated by GLUT5. The internalization of PPN MM and

PPF MM displayed no signiﬁcant differences with and

without free ^d-fructose in L929 cells (p > 0.05, Figure 4B),

3.3. Cellular Uptake Efﬁciency in MCF-7

and RAW264.7 Cells

To investigate the cellular uptake behavior, the internali-

zation of PPN MM and PPF MM by MCF-7 and RAW264.7

cells was analyzed by both ﬂow cytomertry and CLSM. As

shown in Figure 3A, the ﬂuorescence intensity of the mixed

micelles increased dramatically over time. As expected, the

uptake of the two mixed micelles was much higher than

C6 solution (p < 0.05). The uptake of PPF MM was 1.7 times

higher than those treated with PPN MM at 5 min and 2.3

times higher at 4 h (p < 0.05) in MCF-7 cells. These results

may be attributed to the afﬁnity between GLUT family

and the presence of ^d-fructose on the surface of PPF MM.

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Figure 3. Cellular uptake of PPN MM and PPF MM. Cellular uptake of Coumarin (C6) loaded micelles in A) MCF-7 cells and B) RAW264.7

cells. C) Confocal laser scanning microscopy (CLSM) images of cellular uptake in MCF-7 cells. Scale bar represents 20 µm. *p < 0.05 versus

C6 solution; & p < 0.05 versus PPN MM.

suggesting that PPF MM internalization in L929 cells was

not mediated through GLUT5, but likely through other

transporters/receptors such as GLUT2.^[12,31]Also, PPF MM

showed higher cellular uptake on L929 cells than PPN

MM (Figure S17, Supporting Information), which could

be due to other pathways involved in the internalization

of PPF MM. Together, these results revealed that GLUT5

recognizes ^d-fructose located on the surface of PPF MM

thus inducing GLUT5-mediated internalization of PPF

MM. However, the presence of free ^d-fructose cannot

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Figure 4. Cell internalization pathways of PPN MM and PPF MM. Inhibition of cellular uptake by different concentrations of d-fructose in

A) MCF-7 and B) L929 cells. Mechanistic study of C) PPN MM and D) PPF MM internalization pathways in MCF-7 cells. MCF-7 cells were

preincubated under various conditions including 4 °C, amiloride, chlorpromazine, nystatin, M-β-CD, and sucrose. *p < 0.05. **p < 0.01.

completely inhibit the internalization of PPF MM sug-

gesting some other transporters/receptors such as GLUT2-

mediated pathway be involved in the internalization of

PPF MM.^[12,31]

it in its central nonpolar cavity, and nystatin may bind

to cholesterol and disrupt lipid rafts by directly inserting

into membranes and sequestering cholesterol into com-

plexes.^[33]These evidence may help explain why M-β-CD

inhibited the caveolae-mediated endocytosis while nys-

tatin showed no obvious inhibitory effect. On the other

hand, the intracellular uptake at 37 °C was signiﬁcantly

higher than that at 4 °C (p < 0.05), indicating that energy-

dependent pathway was contributed to the internalization

of PPN MM and PPF MM.

Next, we investigated the intracellular distribution

of mixed micelles in MCF-7 cells (Figure 5). Endosomes

and lysosomes were labeled with LysoTracker red and

C6 loaded mixed micelles which exhibited green ﬂuo-

rescence was used to examine the internalization and

subcellular localization of the mixed micelles in MCF-7

cells in greater detail. Colocalization of mixed micelles

with endosome/lysosome (visible as yellow ﬂuorescence)

was observed 5 min and 2 h after micelles were added to

cells. The overlaps with the lysotracker staining revealed

that the mixed micelles were efﬁciently taken up into

3.4. Internalization Pathways and

Intracellular Distribution

To elucidate internalization pathways of PPF MM and PPN

MM, we examined the inhibitory effect of classic endo-

cytosis inhibitors on the uptake efﬁciency of two mixed

micelles in MCF-7 cells. As shown in Figure 4C,D, M-β-CD

treated group exhibited signiﬁcant uptake reduction com-

pared to control (p < 0.01), while other endocytosis inhibi-

tors showed no obvious impact on the cellular uptake,

which indicated that the cellular uptakes of PPN MM and

PPF MM were both mediated by caveolae-dependent,

clathrin- independent, and macropinocytosis-independent

pathways. Although both M-β-CD and nystatin were

caveolae-mediated endocytosis inhibitors, M-β-CD was

a cholesterol depletion agent, and nystatin inhibited the

endocytosis by cholesterol sequestration effect.^[32]M-β-CD

can selectively extract membrane cholesterol by binding

the endosome/lysosomal compartments, indicating

a

lysosomal pathway after internalization.^[15,25]And both

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PPN MM and PPF MM showed extensive distribution in

the cytoplasm. No obvious changes were observed for

PPN MM and PPF MM, indicating that the conjugation

of PCL-PEG-N₃with 3-O-propinyl-β-d-fructopyranose did

not change the intracellular distribution pattern of the

micelle.

3.5. Penetration of Micelles in MCTS

An in vitro 3D tumor spheroid model of MCF-7 cells

was generated to evaluate the penetration of PPN MM

and PPF MM. By CLSM, we observed penetration proﬁles

of C6 solution and the two micelles at each given time

points (Figure 6A). Both PPF MM and PPN MM showed

increasing levels of penetration into the core of MCTS,

which accumulated over time. In comparison, C6 solu-

tion hardly penetrated into the core of MCTS, and showed

only weak ﬂuorescence in the periphery of the spheroids.

Also, C6-loaded PPF MM showed a more signiﬁcant ﬂuo-

rescence intensity and penetration in MCTS compared to

C6-loaded PPN MM. Semi-quantitative analysis of ﬂuo-

rescence intensity within excised tumor spheroid was

carried out to observe the difference of the ﬂuorescence

Figure 5. Representative images of C6 loaded micelles and

lysotracker in MCF-7 cells at 5 min and 2 h. Scale bar represents 20µm.

Figure 6. Fluorescence distribution of C6 solution and C6 loaded mixed micelles in MCF-7 tumor spheroids. A) Tumor spheroid sections were

observed at given time points at predetermined positions by CLSM. B) Semi-quantitative analysis of ﬂuorescence intensity within excised

tumors over time. Scale bars represent 100 µm.

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Figure 7. A) In vivo imaging of Did loaded micelles in nude mice bearing MCF-7 tumors. 1, saline; 2, Did; 3, PPN MM; 4, PPF MM. Did loaded

micelles were given via tail vein injection at an equivalent dose of 0.015 mg kg⁻¹. B) Ex vivo imaging of excised organs and tumors at 48 h.

intensity among the three groups directly (Figure 6B). Cao

et al. previously demonstrated the presence of TPGS in

the mixed micelles is critical to the penetration efﬁciency

in MCTS.^[15]The excellent penetration proﬁle of PPF MM

may attribute to the presence of the TPGS and the ^d-fruc-

tose. This result was consistent with the cellular uptake

study.

remained strong 48 h after administration, showing pro-

longed circulation of mixed micelles in the systemic cir-

culation in mice. The biodistribution of mixed micelles

in the major organs and tumors after 48 h is shown in

Figure 7B. For Did solution group, liver and spleen showed

strong ﬂuorescence with minimum ﬂuorescence accumu-

lated in the tumor. Compared to PPN MM, less PPF MM

was found in the liver, but speciﬁcally accumulated at the

tumor site, which was consistent with our 2D cell uptake

and 3D tumor spheroid penetration studies. These results

further demonstrated that the presence of ^d-fructose tar-

geting ligands could enhance tumor-speciﬁc accumula-

tion in vivo.

3.6. PPF MM Speciﬁcally Accumulated at the

Tumor Site In Vivo

For Did solution group, no obvious in vivo ﬂuorescence

signal was observed in the tumor bearing mice at the

given time points after intravenous injection. Both PPN

MM and PPF MM showed the time-dependent tumor accu-

mulation behavior (Figure 7A). PPF MM group exhibited

more signiﬁcant accumulation at the tumor site compared

to PPN MM group, indicating PPF MM may have greater

tumor targeting efﬁciency. Also, the ﬂuorescence intensity

4. Conclusion

In sum, d-fructose was introduced to the hydrophilic ter-

minal of the diblock copolymer PCL-PEG via orthogonal

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d-Fructose Modification Enhanced Internalization of Mixed Micelles in Breast Cancer Cells via GLUT5 Transporters

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