A small molecule nanodrug consisting of amphiphilic targeting ligand-chemotherapy drug conjugate for targeted cancer therapy

Article abstract of DOI:10.1016/j.jconrel.2016.03.037

Targeted drug delivery is a broadly applicable approach for cancer therapy. However, the nanocarrier-based targeted delivery system suffers from batch-to-batch variation, quality concerns and carrier-related toxicity issues. Thus, to develop a carrier-free targeted delivery system with nanoscale characteristics is very attractive. Here, a novel targeting small molecule nanodrug self-delivery system consisting of targeting ligand and chemotherapy drug was constructed, which combined the advantages of small molecules and nano-assemblies together and showed excellent targeting ability and long blood circulation time with well-defined structure, high drug loading ratio and on-demand drug release behavior. As a proof-of-concept, lactose (Lac) and doxorubicin (DOX) were chosen as the targeting ligand and chemotherapy drug, respectively. Lac and DOX were conjugated through a pH-responsive hydrazone group. For its intrinsic amphiphilic property, Lac-DOX conjugate could self-assemble into nanoparticles in water. Both in vitro and in vivo assays indicated that Lac-DOX nanoparticles exhibited enhanced anticancer activity and weak side effects. This novel active targeting nanodrug delivery system shows great potential in cancer therapy.

Full text of DOI:10.1016/j.jconrel.2016.03.037

A small molecule nanodrug consisting of amphiphilic targeting lig-
and–chemotherapy drug conjugate for targeted cancer therapy
Quanbing Mou, Yuan Ma, Xinyuan Zhu, Deyue Yan
PII:
DOI:
Reference:
S0168-3659(16)30183-3
doi: 10.1016/j.jconrel.2016.03.037
COREL 8199
To appear in:
Journal of Controlled Release
Received date:
Revised date:
Accepted date:
5 December 2015
1 March 2016
28 March 2016
Please cite this article as: Quanbing Mou, Yuan Ma, Xinyuan Zhu, Deyue Yan,
A small molecule nanodrug consisting of amphiphilic targeting ligand–chemotherapy
drug conjugate for targeted cancer therapy, Journal of Controlled Release (2016), doi:
1
0.1016/j.jconrel.2016.03.037
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A small molecule nanodrug consisting of amphiphilic
targeting ligand-chemotherapy drug conjugate for
targeted cancer therapy
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Quanbing Mou , Yuan Ma , Xinyuan Zhu , and Deyue Yan
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School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites,
Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China.
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Abstract Targeted drug delivery is a broadly applicable approach for cancer therapy. However, the
nanocarrier-based targeted delivery system suffers from batch-to-batch variation, quality concerns and
carrier-related toxicity issues. Thus, to develop a carrier-free targeted delivery system with nanoscale
characteristics is very attractive. Here, a novel targeting small molecule nanodrug self-delivery system
consisting of targeting ligand and chemotherapy drug was constructed, which combined the advantages
of small molecules and nano-assemblies together and showed excellent targeting ability and long blood
circulation time with well-defined structure, high drug loading ratio and on-demand drug release
behavior. As a proof-of-concept, lactose (Lac) and doxorubicin (DOX) were chosen as the targeting
ligand and chemotherapy drug, respectively. Lac and DOX were conjugated through a pH-responsive
hydrazone group. For its intrinsic amphiphilic property, Lac-DOX conjugate could self-assemble into
nanoparticles in water. Both in vitro and in vivo assays indicated that Lac-DOX nanoparticles exhibited
enhanced anticancer activity and weak side effects. This novel active targeting nanodrug delivery
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system shows great potential in cancer therapy.
Graphical abstract
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Keywords: Cancer therapy; Targeted delivery; Small molecule nanodrugs; Amphiphilic self-assembly
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1. Introduction
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Targeted cancer therapy has expanded tremendously in recent years. Owing to the rapid development of
nanotechnology, various active targeting nanocarriers based on the modification of nanoparticles with
tumor-specific molecules have been carried out. These active targeting ligands including antibodies,
peptides and small molecules (e.g., folate [1], galactose [2], lactose [3]) can greatly enhance the cellular
uptake of nanoparticles via receptor-mediated endocytosis. Nanocarriers used for cancer therapies such
as liposomes [4-6], micelles [7-20], protein nanoparticles [21, 22], metallic nanoparticles [23-27],
inorganic nanoparticles [28, 29] can passively accumulate in the tumor site through the enhanced
permeability and retention (EPR) effect [30-34]. Both passive and active targeting properties endow the
delivery systems with enhanced therapeutic activity. Nevertheless, the fabrication of active targeting
nanocarriers is extremely complicated, including materials synthesis/assembly, ligand coupling,
separation and purification, which could cause batch-to-batch variation and quality concerns [35].
Besides, the degradation, metabolism, and excretion of nanocarriers can cause significant toxicity issues
[36] (e.g., mitochondrial damage, cardiovascular effects, platelet aggregation, oxidative stress,
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inflammation). The active targeting nano drug delivery systems with high dose use of nanocarriers do
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increase the therapeutic efficiency, but nanocarriers used are potential hazards. Thus, there are only a
few targeted nanodrug delivery systems in clinical trials [37]. It is an urgent demand to develop a
carrier-free system with excellent targeting ability and weak side effects.
Different from the carrier-based targeted delivery systems, a carrier-free amphiphilic drug-drug
conjugate (ADDC) self-delivery system from direct conjugation of hydrophobic anticancer drug with
hydrophilic anticancer drug has been successfully developed in our group [38]. Inspired by the simple
concept only using the amphiphilic small molecules to form micells, here we develop an active targeting
small molecule nanodrug consisting of the hydrophilic targeting ligand and the hydrophobic
chemotherapy drug through a bio-responsive linkage. This novel active targeting self-delivery system
brings together the antigen-targeting specificity of the targeting ligand, favorable pharmacokinetics of
nanomedicine and the cytotoxic potency of promising chemotherapeutic drugs. Chemotherapeutic drugs
can be released in stimuli circumstance. The drug loading ratio of this active targeting self-delivery
system conjugates is high, for the molecular weights of targeting ligands and anticancer drugs are in the
same order of magnitude. Besides, the toxic anticancer drug moieties of the conjugates are aggregated in
the core of the micelles, and surrounded by the nontoxic targeting ligands, which leads to significant
reduction of the cytotoxicity to normal cells. Moreover, the active targeting self-delivery system can
accumulate at the tumor site both passively and actively.
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As a proof-of-concept, lactose (a small molecular weight hydrophilic hepatocyte targeting molecule,
Lac) [39-41] and doxorubicin (a hydrophobic anticancer drug, DOX) are simply conjugated via a
pH-responsive hydrazone group [42-45] (Lac-DOX) to form an active targeting nanodrug delivery
system, as illustrated in Scheme 1. Similar to ADDC, Lac-DOX conjugate is expected to show favorable
pharmacokinetics and benefits of highly potent anticancer drugs. The structure of Lac-DOX conjugate is
well-defined with drug loading ratio of 61.7%. Lac-DOX conjugate self-assembles into nanoparticles
for its intrinsic amphiphilic property. Self-assembled Lac-DOX nanoparticles can passively accumulate
in the tumor site through EPR effect. More importantly, Lac can greatly enhance the cellular uptake of
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nanoparticles via receptor-mediated endocytosis. Both passive and active targeting properties endow the
delivery system with enhanced therapeutic activity.
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Scheme 1. Lac-DOX Nanoparticles for Passive and Active Targeting Drug Delivery. (a) Schematic
illustration for the self-assembly of amphiphilic Lac-DOX. (b) Lac-DOX nanoparticles
accumulated at the tumor site through both passive and active targeting mechanisms. (c) Free
DOX was released from Lac-DOX nanoparticles to inhibit the proliferation of SMMC-7721 cells.
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2. Materials and methods
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2.1 Materials
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Dimethylsulfoxide (DMSO) was dried over calcium hydride for 48 h and then distilled before use.
Methanol was purchased from Hipure Chem and was high performance liquid chromatography (HPLC)
grade.
3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl
tetrazolium
bromide
(MTT,
Sigma),
4-O-beta-D-galactopyranosyl-D-gluconic acid (Lactobionic acid, 97%, Aladdin), hydrazine hydrate
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(98%, Aladdin), and delta-gluconolactone (99%, Adamas) were used as received. Doxorubicin
hydrochloride (DOX·HCl) was purchased from Beijing Huafeng United Technology Corporation.
2.2 Measurements
Nuclear magnetic resonance (NMR) spectroscopy spectra were performed on a Varian Mercury Plus
400 MHz spectrometer with deuterium oxide (D O) or dimethylsulfoxide-d6 (DMSO-d6) as solvents.
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Ultraviolet-visible absorption (UV-vis) spectra were collected on an Evolution 300 UV-vis
spectrophotometer. Fourier transform infrared spectra (FT-IR) spectra were measured on a Paragon
1,000 instrument. High-resolution mass spectra (HRMS) were recorded on a Waters-ACQUITYTM
UPLC & Q-TOF-MS premier. The fluorescent spectra of sample solutions were performed on a
Perkin-Elmer LS-50B fluorescence spectrometer. The excitation wavelength was set at 488 nm, which
was chosen according to the maximum intensity obtained in the excitation spectra. Step increment was
set as 2 nm, and scan speed was set at 480 nm/min. Dynamic light scattering (DLS) measurements were
performed on a Malvern Zetasizer NanoS apparatus (Malvern Instruments, Ltd.) equipped with a 4 mW
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laser light operating at λ = 633 nm. All samples were measured at a scattering angle of 90 . The
morphology and size of nanoparticles were characterized by a Tecnai G2 Spirit Biotwin instrument at
voltage of 120 kV.
2.3 Synthesis of lactobionolactone
According to the literature procedure [46] with a little modification, lactobionic acid (5.0000 g, 13.96
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mmol) was dissolved in anhydrous methanol (70.0 mL) at 75 C followed by vacuum distillation at 40
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C. The procedure was repeated until lactobionic acid was fully converted to lactobionolactone. H
NMR (400 MHz, DMSO-d ) δ (ppm): 5.33-4.01 (br, OH), 4.34-4.13 (m, 2H, CH), 4.02-3.86 (m, 1H,
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CH), 3.75-3.25 (m, 10H, CH & CH ). C NMR (100 MHz, DMSO-d ) δ (ppm): 173.44, 105.42, 84.19,
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76.01, 73.66, 71.83, 71.63, 71.41, 70.94, 68.55, 62.64, 60.79. HRMS: (ESI) [M-H] calcd. for
C H O , 339.0927, found 339.0927.
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2.4
Synthesis
of
(2R,3R,4R,5R)-2,3,5,6-tetrahydroxy-4-(((2S,3R,4S,
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5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)
oxy)hexanehydrazide
(Lac-NHNH2)
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Lactobionolactone (3.0000 g, 8.82 mmol) was dissolved in anhydrous methanol (40.0 mL) at 25 C.
Hydrazine hydrate (2.2270 g, 44.10 mmol) was added dropwise to the solution of lactobionolactone via
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a syringe and keep at 25 C for 1 h. The white powder was collected by a rotary evaporator (yield:
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76.2%). H NMR (400 MHz, DMSO-d ) δ (ppm): 8.91-8.50 (s, 1H, NH), 5.23-4.03 (br, OH), 4.32-4.17
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(d, J = 4.27 Hz, 1H, CH), 4.19-4.09 (d, J = 4.16 Hz, 1H, CH), 4.04-3.93 (m, 1H, CH), 3.73-3.60 (m, 2H,
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CH), 3.60-3.46 (m, 5H, CH & CH ), 3.45-3.24 (m, 3H, CH), 3.18-3.16 (s, 2H, NH ). C NMR (100
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MHz, DMSO-d ) δ (ppm): 171.46, 104.95, 82.96, 76.15, 73.67, 72.05, 71.86, 71.56, 70.92, 68.74,
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62.77, 61.15. HRMS: (ESI) [M+H] calcd. for C H O , 339.0927, found 339.0927.
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2.5
Synthesis
of
(2R,3R,4R,5R,Z)-N'-(1-(4-(((2R,4S,5S,6S)-4-amino-
5-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-1,2,3,
4,6,11-hexahydrotetracen-2-yl)-2-hydroxyethylidene)-2,3,5,6-tetrahydroxy-4-(((2S,3R,4S,5R,6R)-3
,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexanehydrazide
(Lac-DOX)
Doxorubicin hydrochloride (500.0 mg, 0.86 mmol) was dissolved in anhydrous DMSO (10.0 mL).
Lac-NHNH (643.8 mg, 1.73 mmol) in 10.0 mL DMSO was added dropwise to the above solution. The
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resulting solution was stirred at 25 C for 0.5 h under an atmosphere of nitrogen. After evaporation of
DMSO under reduced pressure, the resulting mixture was purified by reverse phase silica gel column
chromatography (gradient 2 to 20 percent acetonitrile in water) to give the title compound (250.0 mg,
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32.4%). H NMR (400 MHz, DMSO-d ) δ (ppm): 11.68 (s, 1H, NH), 8.02 (br, 2H, NH ), 7.90-7.76 (m,
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2H, CH), 7.64-7.53 (d, J = 7.59 Hz, 1H, CH), 6.15-5.96 (br, OH), 5.65-4.42 (br, OH), 5.35-5.26 (m, 1H,
CH), 5.08-5.01 (m, 1H, CH), 4.48 (s, 2H, CH ), 4.28 (m, 1H, CH), 4.24 (m, 1H, CH), 4.16 (m, 1H, CH),
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4.14 (m, 1H, CH), 3.95 (s, 3H, CH ), 3.71 (m, 1H, CH), 3.70 (m, 1H, CH), 3.61 (m, 1H, CH), 3.58 (m,
3
1H, CH), 3.55 (m, 2H, CH ), 3.46 (m, 2H, CH ), 3.39 (m, 1H, CH), 3.37 (m, 1H, CH), 3.35 (m, 1H,
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CH), 3.30 (m, 1H, CH), 2.94 (m, 2H, CH ), 2.22 (m, 2H, CH ), 1.96-1.59 (m, 2H, CH ), 1.26-1.12 (d, J
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= 1.15 Hz, 3H, CH ). C NMR (100 MHz, DMSO-d ) δ (ppm): 186.71, 186.63, 169.06, 161.13,
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158.53, 156.75, 155.13, 136.69, 136.20, 135.57, 134.95, 120.17 (overlap, 2C), 119.34, 110.82 (overlap,
2C), 105.18, 99.76, 84.01, 76.13, 73.70, 72.82, 72.10, 71.60 (overlap, 3C), 71.04, 68.57, 66.65, 66.58,
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62.66, 60.93, 57.78, 57.04, 47.06, 38.33, 33.37, 28.73, 17.30. HRMS: (ESI) [M+H] calcd. for
C H N O , 898.3093, found 898.3091.
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2.6 Synthesis of (2R,3R,4R,5S)-2,3,4,5,6-pentahydroxyhexane hydrazide (Glu-NHNH2)
Hydrazine hydrate (7.0270 g, 140.40 mmol) was added dropwise to a solution of delta-gluconolactone
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(5.0000 g, 28.08 mmol) in methanol (80.0 mL) under constant stirring at 40 C for 0.5 h. The white
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powder was collected by vacuum filtration (yield: 60.0%). H NMR (400 MHz, DMSO-d ) δ (ppm):
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8.80 (s, 1H, NH), 5.26 (s, 1H, OH), 4.30-4.70 (br, 4H, OH), 4.22 (s, 2H, NH ), 4.07-3.98 (m, 1H, CH),
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3.92-3.83 (m, 1H, CH), 3.62-3.31 (m, 4H, CH & CH ). C NMR (100 MHz, DMSO-d ) δ (ppm):
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171.63, 73.52, 72.35, 71.89, 70.62, 63.82. HRMS: (ESI) [M+H] calcd. for C H N O , 211.0930, found
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211.0935.
2.7
Synthesis
of
(2R,3R,4R,5S)-N'-(1-(4-(((2R,4S,5S,6S)-4-amino-5-
hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-1,2,3,4,
6,11-hexahydrotetracen-2-yl)-2-hydroxyethylidene)-2,3,4,5,6-pentahydroxyhexanehydrazide
(Glu-DOX)
Glu-NHNH (361.4 mg, 1.72 mmol) in anhydrous DMSO (10.0 mL) was added dropwise to a solution
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of doxorubicin hydrochloride (500.0 mg, 0.86 mmol). The resulting solution was stirred at 25 C for 0.5
h under an atmosphere of nitrogen. After evaporation of DMSO under reduced pressure, the resulting
mixture was purified by reverse phase silica gel column chromatography (gradient 2 to 20 percent
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acetonitrile in water) to give the title compound (280.0 mg, 44.0%). H NMR (400 MHz, DMSO-d ) δ
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(ppm): 11.63 (s, 1H, NH), 8.02 (br, NH ), 7.73-7.88 (m, 2H, CH), 7.62-7.51 (d, J = 7.58 Hz, 1H, CH),
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6.07-5.94 (br, OH), 5.78-5.65 (br, OH), 5.56-5.36 (br, OH), 5.37-5.21 (m, 1H, CH), 5.18-5.09 (br, OH),
5.09-4.98 (m, 1H, CH), 4.76-4.29 (br, CH & OH), 4.24-4.08 (m, 2H, CH), 4.07-3.80 (m, 4H, CH &
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CH ), 3.66-3.22 (m, 6H, CH & CH ), 3.09-2.86 (m, 2H, CH ), 2.35-2.01 (m, 2H, CH ), 1.96-1.58 (br,
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2H, CH ), 1.26-1.14 (d, J = 1.19 Hz, 3H, CH ). C NMR (100 MHz, DMSO-d ) δ (ppm): 186.63
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(overlap, 2C), 169.16, 161.14, 158.56, 156.76, 155.15, 136.65, 136.21, 135.59, 134.98, 120.13 (overlap,
2C), 119.33, 110.83 (overlap, 2C), 99.78, 74.25, 72.79, 72.15, 72.02, 71.59, 70.56, 66.68, 66.60, 63.77,
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57.72, 57.04, 47.07, 38.36, 33.40, 28.78, 17.30. HRMS: (ESI) [M+H] calcd. for C H N O ,
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736.2565, found 736.2555.
2.8 Preparation of Lac-DOX and Glu-DOX nanoparticles
Briefly, Lac-DOX (5.0 mg) was dissolved in 5.0 mL methanol. The solution was evaporated in a
pear-shaped flask under reduced pressure. Ultra pure water (5.0 mL) was added to the flask under
ultrasonic field to produce a solution with a concentration of 1.0 mg/mL for further experiments.
Glu-DOX nanoparticles were obtained following the same procedure.
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2.9 Critical aggregation concentration (CAC) measurements
To investigate the aggregation of Lac-DOX, the CAC measurements were performed. Nile red in
acetone was added to indicate concentrations of Lac-DOX in water and the concentration of nile red was
fixed at 10 μM. The fluorescence emission intensity of wavelength at 620 nm was determined by means
of excitation wavelength at 543 nm.
2.10 Preparation of Cy5.5-loaded Lac-DOX nanoparticles
4.0 mg Lac-DOX and 0.4 mg Cy5.5 were dissolved in 2.0 mL of DMSO. Then the mixture was added
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dropwise to 2.0 mL ultra pure water and stirred at 25 C for 15 min. Subsequently the solution was
dialyzed against ultra pure water for 12 h (molecular weight cutoff (MWCO) = 1,000 g/mol) and the
ultra pure water was exchanged every 3 h. To determine the content of Cy5.5 in Lac-DOX
nanoparticles, the solution was lyophilized and then redissolved in DMSO. The amount of Cy5.5 in
Lac-DOX nanoparticles was determined by measuring the absorbance at 680 nm with a UV/Vis
spectrophotometer, wherein a calibration curve was obtained with Cy5.5/DMSO solutions with different
Cy5.5 concentrations.
2.11 In vitro acid-triggered drug release study
The release profiles of doxorubicin from Lac-DOX nanoparticles were studied at pH 5.5 and pH 7.4 in
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dialysis tubes with a MWCO of 3000 at 37 C. At desired time intervals, 0.5 mL buffer solution
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outside the dialysis bag was taken out for HPLC measurements and replenished with an equal volume of
fresh media to keep the sink condition. The amount of doxorubicin was determined by an HPLC Agilent
1260 Infinity system (Agilent) using a 3/1 (V/V) mixture of methanol and water as a mobile phase.
2.12 Cell culture
SMMC-7721 cells (a human hepatocellular carcinoma cell line) were cultured in Dulbecco's Modified
Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics (50 units/mL
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penicillin and 50 units/mL streptomycin) at 37 C in a humidified atmosphere containing 5% CO .
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2.13 Cellular uptake
Cellular uptake by SMMC-7721 cells was performed on flow cytometry and confocal laser scanning
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microscope (CLSM), respectively. For flow cytometry studies, approximately 5 × 10 SMMC-7721
cells were seeded in 6-well culture plates per well and cultured overnight, followed by removing culture
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medium and adding Lac-DOX nanoparticles (equivalent DOX concentration: 8 μg/mL) at 37 C for
predetermined time intervals. Thereafter, drug solutions were removed and the cells were washed three
times with PBS and treated with trypsin. The cells were detached from cell culture. Then the solutions
were centrifugated for 5 min (1000 rpm). After the supernates were removed, the cells were
resuspended in 0.5 mL of PBS. A single cell suspension was prepared by filtration through a 300 mesh
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filter. Finally, data for 1 × 10 gated events were collected by means of a flow cytometry on a
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LSRFortessa (BD) and analyzed by BD FACSDiva software. For CLSM studies, SMMC-7721 cells
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were seeded in 12-well culture plates (a clean coverslip was put in each well) at 2 × 10 cells per well in
1 mL of complete DMEM and grown overnight, followed by removing culture medium and adding
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Lac-DOX nanoparticles (equivalent DOX concentration: 8 μg/mL) at 37 C for predetermined time
intervals. The supernatant was carefully removed and the cells were washed with ice-cold PBS three
times. Subsequently, the cells were fixed with 4% formaldehyde in each well for 30 min at room
temperature and washed three times with ice-cold PBS again. Finally, Hoechst 33342 fluorescence dye
was added to every sample for 15 min to stain the cell nuclei. The slides were rinsed with PBS for three
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times. The slides were mounted and observed by a Nikon Ti-E inverted motorized microscope with a
Nikon A1Si spectral detector confocal system running NIS-C Elements software.
2.14 In vitro cell proliferation assay
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SMMC-7721 cells were plated in 96-well plates at 8 × 10 cells per well and further incubated for 24 h.
The medium was removed and serial dilutions of DOX·HCl or Lac-DOX nanoparticles were added to
cells. The cells were grown for another 48 h. Then, MTT was used for mitochondrial activity evaluation
in cell viability studies. The medium was removed carefully after the cells were incubated for 4 h. The
obtained blue formazan crystals were dissolved in 200 μL DMSO per well, and the absorbance was
measured in a Perkin-Elmer 1420 Multilabel counter at a wavelength of 490 nm.
2.15 Cell apoptosis assay
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Apoptosis in cells was visualized using an Alexa Fluor 488 annexin V/Dead cell apoptosis kit
according to the manufacturer’s protocol. Briefly, SMMC-7721 cells were plated in 6-well plates at 5 ×
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10 cells per well and further incubated for 24 h. Apoptosis in cells was induced by treating cells with
DOX·HCl or Lac-DOX nanoparticles at equivalent DOX concentration (2 μg/mL) for 12 h. A negative
control was prepared by incubating cells in the absence of inducing agent. Cells were harvested, washed
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twice with cold PBS and stained with Alexa Fluor 488 annexin V and PI according to the
manufacturer’s instructions. The stained cells were analyzed by a flow cytometry on a LSRFortessa
(BD).
2.16 Animals
All male Balb/c nude mice (18-20 g) and SD rats (~200 g) were purchased from Chinese Academy of
Sciences (Shanghai, China). Animal studies were conducted in accordance with the guidelines for the
care and use of laboratory animals proved by the Animal Ethics Committee of Shanghai Jiao Tong
University. SMMC-7721 cells were injected subcutaneously in flanks of male Balb/c nude mice at 2 ×
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10 cells per tumor. The tumors were allowed to grow for around 50 mm and 250 mm for antitumor
studies and in vivo optical imaging experiments, respectively.
2.17 In vivo antitumor studies
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The tumor bearing nude mice as described above were randomly divided into three cohorts of six mice
each. Lac-DOX nanoparticles and free DOX·HCl were intravenously injected via the tail vein at a dose
of 10 mg/kg (200 μL) every three days for 27 days. The control group only received saline (200 μL).
The width and length of the tumors and the body weight of mice were measured at the time of each
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treatment. Tumor volume (V) was calculated using the following equation: V (mm ) = 1/2 × length
(mm) × width (mm) × width (mm). One mouse in each group was sacrificed on day 18. Tumors as well
as major organs (heart, liver, spleen, lung and kidney) were dissected, weighted, sectioned into around 3
mm slices and fixed in 4% paraformaldehyde overnight for histology and immunohistochemical
analysis.
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2.18 Histology and immunohistochemical analyses
For histology analysis, tumors and major organs were treated according to the routine histological
procedures. Briefly, they were embedded in paraffin. The paraffin embedded tumors and organs were
cut at 5 μm thickness. Sections were subsequently stained with hematoxylin solution and eosin Y
solution (H & E) to assess histological alterations by microscope (Nikon ECLIPSE Ti). For
immunohistochemical analysis, the paraffin-embedded 5-µm-thick tumor sections were mounted on
glass slides. Tissue sections were deparaffinized in xylene, rehydrated in graded alcohols (100%, 95%,
85%, 75%) and washed in distilled water. Endogenous peroxidase was blocked by 3% hydrogen
peroxide aqueous solution for 10 min, and the slides were heated to boil in EDTA Antigen Retrieval
Solution for 10 min by using water bath for antigen retrieval. Thereafter, the sections were cooled,
rinsed twice with PBS. Non-specific bonding sites were blocked with goat serum in TBS for 20 min.
The sections were incubated for 1 h with PCNA antibody (1:100) at room temperature. Then, the slides
were rinsed three times with PBS and incubated for 1 h with a goat anti-mouse HRP secondary antibody
at a dilution of 1:1000 followed by colorimetric detection using DAB. Finally, the sections were
counterstained with hematoxylin and prepared for mounting. Images were taken on a Nikon ECLIPSE
Ti microscope.
2.19 In vivo optical imaging and ex vivo biodistribution studies
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In vivo optical imaging was conducted and analyzed using a Kodak multimode imaging system with
690 nm excitation wavelength and 700 nm emission wavelength. The tumor bearing nude mice were
anesthetized with 4% chloral hydrate. Cy5.5-loaded Lac-DOX nanoparticles and free Cy5.5 were
injected through the vein tail and imaging was performed at 1, 2, 3, 4 and 8 h postinjection. For the ex
vivo biodistribution study, the tumor bearing nude mice were injected with Lac-DOX nanoparticles and
free DOX·HCl (10 mg/kg bw).Tumor tissues and major organs were carefully harvested at 1, 2 and 8 h
postinjection. All samples were rinsed with saline and immediately imaged using a Kodak multimode
imaging system.
2.20 Pharmacokinetics
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To determine the pharmacokinetics profiles of Lac-DOX nanoparticles and free DOX·HCl, SD rats
(~200 g) were randomly divided into 2 groups (n = 4). Drugs were intravenously injected through tail
vein at a dose of 10 mg/kg. The blood samples (0.2 mL) were collected via eye puncture at the time
point of 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h. Blood samples were processed for
serum by centrifugation (10 min, 1000 rpm) to determine the pharmacokinetics property of Lac-DOX
nanoparticles and free DOX·HCl.
2.21 Blood biochemistry assay
The tumor bearing nude mice were intravenously injected via the tail vein at a dose of 10 mg/kg (200
μL) every three days for 15 days. The blood samples were collected via eye puncture for blood
biochemistry assay.
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3. Results and discussions
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3.1 Synthesis of Lac-DOX and Preparation of Lac-DOX nanoparticles
The synthesis and self-assembly of Lac-DOX are showed in Scheme 2, and the details are described
in Methods. Briefly, lactobionolactone was first synthesized via the lactonization of lactobionic acid.
Lactobionolactone was then converted to Lac-NHNH by hydrazinolysis. Lac-NHNH reacted with
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doxorubicin hydrochloride (DOX•HCl) to produce Lac-DOX. The structure of Lac-DOX was confirmed
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by H and C NMR. As shown in Fig. 1, the proton signal at 11.68 ppm (1) belongs to the secondary
amine of the acylhydrazone group and the proton signals at 6.15-5.96 ppm and 5.65-4.42 ppm belong to
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the hydroxyl protons of Lac in the H NMR spectrum of Lac-DOX. Besides, the carbon signal at 214.57
ppm (2) corresponding to the carbonyl group of DOX disappears, and a new peak appears at 158.53
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ppm (2’) corresponding to R R C=NNH- group of Lac-DOX in the C NMR spectrum. Lac-DOX was
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also characterized by ultraviolet/visible spectrophotometer (UV/Vis), Fourier transform infrared
spectroscopy (FTIR), fluorescence spectroscopy (Figs. S3-S5) and high-resolution mass spectra
(HRMS). All experimental results demonstrate that Lac-DOX has been synthesized successfully. For
comparison, delta-gluconolactone doxorubicin conjugate (Glu-DOX) was synthesized using the method
of two steps (Fig. S1) by reaction of hydrazine hydrate with delta-gluconolactone to synthesize
Glu-NHNH and then by reaction of Glu-NHNH with DOX•HCl to produce Glu-DOX (Fig. S2).
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Scheme 2. Synthesis Route of Lac-DOX and Schematic Representation of the Self-Assembled
Lac-DOX Nanoparticles.
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Fig. 1. H NMR and C NMR spectra of DOX and Lac-DOX. 1: the proton signal of the
secondary amine of the acylhydrazone group (Lac-DOX), 2: the carbon signal of the carbonyl
group (DOX), 2’: the carbon signal of R R C=NNH- group (Lac-DOX). (Note: The signal of the
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C NMR spectra around 40 ppm is the solvent peak of DMSO.)
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Due to its amphiphilicity, Lac-DOX self-assembled into nanoparticles in water. As shown in Fig. 2a,
the average hydrodynamic diameter of Lac-DOX nanoparticles is about 172.8 nm determined by DLS.
The morphology of the nanoparticles was observed by transmission electron microscopy (TEM). The
average diameter of these spherical nanoparticles consisting of a lot of small spherical domains is
approximate 150 nm from the TEM image in Fig. 2b, which is consistent with the DLS results. We infer
that these nanoparticles are a type of multi-micelles aggregates (MMA), which has already been well
reported [38, 47]. The CAC measurements were performed to investigate the self-assembly behavior of
Lac-DOX by using Nile Red as a fluorescent probe. Nile Red does not emit fluorescence in aqueous
solution, but its fluorescence is known to increase substantially in the hydrophobic environments of
micelles [48-51]. The fluorescence intensities were monitored by adding different amounts of Lac-DOX
into an aqueous dispersion of Nile Red (10 μM). Fig. S7 shows the observed inflection point
corresponding to a CAC of 2 μg/mL, indicating the rather high stability of Lac-DOX nano-particles
[52].
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Fig. 2. (a) DLS profile of Lac-DOX nanoparticles, the average size (Dh = 172.8 nm) and the
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polydispersity index (PDI = 0.173). (b) TEM image of Lac-DOX nanoparticles. Scale bars: 200
nm.
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3.2 In Vitro Drug Release
As a pH-responsive molecule, Lac-DOX could be hydrolyzed to lactobionic acid and DOX at an
acidic pH. Lac-DOX nanoparticles were incubated under different pH conditions (pH 7.4 and 5.5)
corresponding to the pH of blood and late endosome [53]. An aliquot of the Lac-DOX nanoparticles was
taken out for HPLC measurements at predetermined time intervals. As shown in Fig. S6, almost all of
DOX is released at pH 5.5 after 24 h incubation, while little DOX is released within 24 h at pH 7.4. The
release profiles indicate that Lac-DOX nanoparticles remain stable in the blood circulation and normal
extracellular environment, while DOX can be rapidly released under endo/lysosomes conditions of
cancer cells.
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3.3 In Vitro Studies of Lac-DOX Nanoparticles
To investigate the proliferation inhibition of SMMC-7721 cells (a human hepatocellular carcinoma
cell line) induced by Lac-DOX nanoparticles and free DOX, MTT assay was used and the details are
described in the Supporting Information. Fig. 3a shows that the cytotoxicity of Lac-DOX to
SMMC-7721 cells is nearly the same as that of free DOX, indicating that Lac-DOX and free DOX
almost have the same anticancer ability in vitro. It can be attributed to the fast release of free DOX from
the Lac-DOX nanoparticles (Fig. S6).
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Fig. 3. (a) Cell viability of SMMC-7721 cells against Lac-DOX nanoparticles or DOX after culture
for 48 h. (b) In vitro cellular uptake studies of Lac-DOX nanoparticles in SMMC-7721 cells
determined by FCM. (c) Apoptosis was evaluated following treatment of SMMC-7721 cells
induced by DOX or Lac-DOX nanoparticles at equivalent DOX concentration (2 μg/mL) for 12 h,
by staining with Annexin V-FITC & PI. Early apoptotic cells are presented in the Lower right
quadrant, and late apoptotic cells are presented in the upper right quadrant.
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Cellular uptake behaviors of Lac-DOX nanoparticles were investigated by FCM and CLSM against
SMMC-7721 cells. Fig. 3b shows the mean fluorescence intensity of the aromatic chromophore of DOX
after incubation with Lac-DOX nanoparticles for 15, 30, 45 and 60 min. The intracellular DOX
fluorescence increases with increasing incubation time, which can be attributed to the cellular uptake of
Lac-DOX nanoparticles by SMMC-7721 cells. The outcome of CLSM (Fig. S10) is consistent with the
FCM results. The nucleus region was stained into blue by Hoechst 33342. Here, the red fluorescence is
emitted from free DOX or Lac-DOX. As shown in Fig. S10, free DOX, a small molecular anticancer
drug, can rapidly diffuse into the nucleus regions of SMMC-7721 cells within 15 min. While for
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Lac-DOX nanoparticles, the red fluorescence of nucleus region gradually increases with the
accumulation of DOX from Lac-DOX nanoparticles.
As a fundamental biological phenomenon, apoptosis plays a crucial role in normal tissue homeostasis.
To induce apoptosis is also a common way to treat cancer [54-56]. For example, many anticancer drugs
(e.g., DOX) kill cancer cells primarily by inducing apoptosis. Annexin V, which has a high affinity for
phosphatidyl serine, labeled with fluorescein isothiocyanate (FITC) can identify apoptotic cells by
binding to phosphatidyl serine exposed on the outer leaflet. Propidium iodide (PI) binding tightly to the
nucleic acids in the cell specifically stains dead cells with red fluorescence. Thus the SMMC-7721 cells
treated by free DOX and Lac-DOX nanoparticles were probed by Annexin V-FITC & PI assay and
analyzed by flow cytometry (FCM) to further quantify the cell apoptosis [57, 58]. As illustrated in Fig.
3c, the percentages of the apoptosis cells are 76.8% and 79.3% in free DOX and Lac-DOX nanoparticle
groups, respectively, which is quite consistent with the result of MTT assay. Both MTT and apoptosis
results indicate that Lac-DOX nanoparticles and free DOX almost have the same capability to inhibit
the growth of SMMC-7721 cells.
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3.4 Passive and active targeting behaviors of Lac-DOX
Galactosyl has a specific interaction with the asialoglycoprotein (ASGP) receptors over-expressed on
hepatocyte membranes [59, 60]. Lac is a disaccharide sugar derived from galactose and glucose. Thus
Lac-DOX nanoparticles are expected to have liver-targeting ability. To evaluate the active-targeting
efficiency of Lac-DOX nanoparticles, galactose-free Glu-DOX nanoparticles were used as control.
SMMC-7721 cells were incubated with Lac-DOX nanoparticle solution and Glu-DOX nanoparticle
solution (equivalent DOX concentration: 8 μg/mL) for predetermined time intervals, respectively. The
mean fluorescence intensity of DOX was determined by FCM. As shown in Fig. 4a, although the
equivalent DOX concentration is used, the SMMC-7221 cells incubated with Lac-DOX nanoparticles
display much higher fluorescence intensity than those with Glu-DOX nanoparticles. It indicates that
Lac-DOX can actively target SMMC-7721 cells by specifically interacting with the ASGP receptors.
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Fig. 4. The active and passive target behaviors of Lac-DOX nanoparticles. (a) Non-liver-targeting
Glu-DOX nanoparticles (the left fig.), and the right fig. showed the time-dependent profiles of
Lac-DOX nanoparticles and Glu-DOX nanoparticles mean fluorescence intensity in SMMC-7721
cells. (b) In vivo imaging of the tumor-bearing nude mice was performed after intravenous
injection of free Cy5.5 or Cy5.5-loaded Lac-DOX nanoparticles. The fluorescence images were
acquired at post-injection with 1 h, 2 h, 3 h, 4 h and 8 h.
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To investigate whether Lac-DOX nanoparticles could passively target tumor sites via the EPR effect,
in vivo optical imaging was conducted. The Cy5.5-loaded Lac-DOX nanoparticles were prepared for
real-time in vivo imaging. These nanoparticles and free Cy5.5 were injected intravenously into the
tumor bearing nude mice. The fluorescence signals of Cy5.5 were recorded on a Kodak multimode
imaging system at 1, 2, 3, 4 and 8 h postinjection. Fig. 4b shows that during the whole time observation,
the fluorescence of free Cy5.5 remains weak at the tumor site, while the fluorescence signal of
Cy5.5-loaded Lac-DOX nanoparticles appears at the tumor site at 1 h postinjection. Even 8 h after
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injection the fluorescence signal still remains strong, confirming the accumulation of Lac-DOX
nanoparticles in the tumor. The accumulation of Lac-DOX nanoparticles can be attributed to the EPR
effect. The nanoparticles with suitable size are trapped due to the tortuous and leaky vasculatures in the
tumor. The cellular uptake behaviors of Lac-DOX nanoparticles against SMMC-7721 cells and in vivo
optical imaging results indicate that Lac-DOX nanoparticles can target liver tumor both actively and
passively.
3.5 In vivo antitumor studies
To investigate the therapeutic efficiency of Lac-DOX nanoparticles in vivo, the athymic nude mice
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bearing human hepatoma SMMC-7721 cells were used. The tumor volume reached about 50 mm in
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one week. Therapeutic details are shown in the Supporting Information. The width and length of the
tumors and the body weight of mice were measured before the time of each treatment. As illustrated in
Fig. 5a, the therapeutic efficiency of free DOX and Lac-DOX nanoparticles is comparable till day 9.
The therapeutic efficiency of Lac-DOX nanoparticles is a little lower than that of free DOX between
day 12 and day 15, which can be attributed to the dramatic decrease in body weight of DOX-treated
group (Fig. 5c), leading to the deceptive therapeutic effect. Owing to severe side effects of DOX
causing the death of DOX-treated group, no therapeutic data of DOX-treated group is recorded after day
15. However, the tumor growth of Lac-DOX nanoparticle treated group is inhibited (Fig. 5a) and the
mean body weight has no significant change compared with the control group (Fig. 5c). These results
demonstrate that the therapeutic effects of free DOX and Lac-DOX nanoparticles are comparable, which
are consistent with the results of experiments. In the meantime, Lac-DOX nanoparticles show weak side
effects on the tumor-bearing nude mice (Figs. 5b and 5c).
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Fig. 5. In vivo therapeutic efficacy of Lac-DOX nanoparticles in the athymic nude mice bearing
human hepatoma SMMC-7721 cells. (a) Tumor volumes of each group were recorded after
intravenous injection of saline, free DOX and Lac-DOX nanoparticles (n = 6 in each group). The
tumor volumes were normalized to their initial sizes. Statistical significance: *P < 0.05, **P < 0.01
and ***P < 0.001. (b) Photographs of the tumor-bearing nude mice after treatment on day 1, day
12 and day15. (c) Body weight changes of the tumor-bearing mice during the experiments. (d)
Survival curves of the tumor-bearing mice with indicated treatments.
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3.6 Pharmacokinetics
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Compared with small molecular drugs, nanoparticles with suitable size exhibit improved
pharmacokinetic properties in the bloodstream. The serum DOX and Lac-DOX concentration-time
profiles are illustrated in Fig. 6c. The concentration of Lac-DOX is much higher than that of free DOX
at the same time up to 10 h. These results show that compared with free DOX the clearance of
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Lac-DOX nanoparticles is slow down due to their nanoscale characteristics in the bloodstream. To
further confirm the above results, the ex vivo biodistribution study, histology analysis,
immunohistochemical analysis and serum biochemistry assays were performed.
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Fig. 6. (a) Microscopic images of H&E-stained sections of the major organs and tumors (on day
18). (b) Immunohistochemical analysis of tumors by PCNA antibody. The tumor tissues were
harvested after 15 days of treatment. (c) In vivo pharmacokinetics profiles of drugs in SD rats (n
= 4 in each group). (d) Images of the major organs and tumors harvested at 1, 2 and 8 h
postinjection. 1 heart, 2 liver, 3 spleen, 4 lung, 5 kidneys, 6 tumor.
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For the ex vivo biodistribution study, tumor tissues and major organs (heart, liver, spleen, lung,
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kidney) were carefully excised at different time intervals and the fluorescence of the aromatic
chromophore of DOX was monitored. As shown in Fig. 6d, Lac-DOX nanoparticles mainly accumulate
in the tumor site during the whole time observed. The fluorescence signals in major organs are relatively
weak, indicating the weak side effects of Lac-DOX nanoparticles. While for free DOX, the fluorescence
signals measured at tumor site become weaker and weaker in a short time. In major organs it mainly
accumulates in the kidney at 1 h postinjection. Then free DOX accumulates in the liver at 2 h
postinjection. At 8 h postinjection the signals of free DOX can still be detected in the liver. The long
time accumulation of free DOX in major organs may cause severe side effects.
3.8 Histology and immunohistochemical analyses
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The accumulation of free DOX in the liver and kidneys might cause some side effects. Thus the
major organs and tumors were harvested on day 18 for histology (Fig. 6a) and immunohistochemical
(Fig. 6b) analyses. The tumors and major organs were treated according to the routine histological
procedures. Comparing with the PBS control group, the major organs of Lac-DOX nanoparticle group
show no tissue damage. While for free DOX-treated group, the spleens and kidneys are severely
damaged. The morphology of tumor cells is obviously changed except for the PBS control group. Then
the proliferating cell nuclear antigen (PCNA) images were used to study the proliferation of tumor cells.
PCNA-positive nuclei were brown, and PCNA-negative nuclei were blue. The nucleus of the PBS
control group was stained by brown. However, most tumor cells of free DOX group and Lac-DOX
nanoparticle group were blue. These results are quite compatible with that of in vivo antitumor studies.
That is to say Lac-DOX nanoparticles show comparable antitumor activity compared with free DOX.
However, no obviously side effects are observed for Lac-DOX nano-particle group.
3.9 Blood biochemistry assay
Besides, the blood biochemistry indexes were also measured to assess the toxicities from DOX. As
shown in Fig. 7, blood urea nitrogen (Urea) and creatinine (Cr) of Lac-DOX nanoparticle group do not
show significant difference with control group. But for free DOX group, the Urea value is obviously
lower than that of control group, indicating that obvious hepatic or kidney dis-order[61] is induced by
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free DOX.
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Fig. 7. Blood biochemical indexes of SMMC-7721 tumor-bearing nude mice after free DOX and
Lac-DOX nanoparticle treatments. Mean and standard deviation of blood urea nitrogen (Urea)
and creatinine (Cr) of SMMC-7721 tumor-bearing nude mice (n = 5).
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4. Conclusions
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ADDC combines the advantages of highly potent anticancer drugs and long blood circulation time
together without carriers. However, the toxic anticancer drug moieties of ADDC are aggregated not only
in the core but also in the surface of the micelles, which may lead to undesired toxicities during the
blood circulation. Besides, the targeted ability of ADDC is limited only through EPR effect. Making use
of the advantages and bypassing some disadvantages of ADDC, here we have developed a novel kind of
active targeting nanodrug delivery system, which is fabricated via conjugating targeting ligand such as
Lac to chemotherapy drug such as DOX by a facile way with on-demand drug release behavior.
Different from nanocarrier-based drug delivery systems, the annoying problems from the degradation,
metabolism and excretion of carriers can be completely eliminated in our system. For Lac-DOX
conjugate, the drug loading efficiency is 61.7%, which is tremendously high. Benefiting from its
amphiphilicity, Lac-DOX conjugate self-assembles into nanoparticles with suitable size. At an
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endosome/lysosome acidic environment, DOX is completely released from Lac-DOX nanoparticles
within 24 h. The anticancer activity of Lac-DOX nanoparticles is assessed both in vitro and in vivo. The
cell proliferation assay and apoptosis assay indicate that Lac-DOX nanoparticles and free DOX almost
have the same capability to inhibit the growth of SMMC-7721 cells. In addition, due to their improved
pharmacokinetic properties and passive and active tumor targeting abilities, Lac-DOX nanoparticles
mainly accumulate at the tumor site with enhanced cellular uptake. Besides, the lower uptake of
Lac-DOX in major organs, normal body weights and normal blood biochemistry indexes indicate that
Lac-DOX nanoparticles show very weak side effects in vivo. Consequently, this kind of active targeting
nanodrug delivery system consisting of amphiphilic targeting ligand-chemotherapy drug conjugates
would open the doors to a new strategy for cancer therapy.
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Acknowledgments
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The authors thank the National Basic Research Program (2015CB931801) and the National Natural
Science Foundation of China (51473093). We thank Lei Feng (Instrumental Analysis Center of
Shanghai Jiao Tong University) for HRMS assay, Yue Su and Linzhu Zhou for in vitro experiments and
Bing Liu and Dali Wang for in vivo experiments. We are also grateful to Mo Sun for an enlightening
discussion.
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Author information
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Corresponding authors: Tel.: +86-21-54742664; Fax: +86-21-54741297; Correspondence and
requests for materials should be addressed to D. Y. (dyyan@sjtu.edu.cn) or to X. Z.
(xyzhu@sjtu.edu.cn). The authors declare no competing financial interests.
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Both authors contributed equally to this manuscript.
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