A multi-functional hypoxia/esterase dual stimulus responsive and hyaluronic acid-based nanomicelle for targeting delivery of chloroethylnitrosouea | Journal of Nanobiotechnology


3-Aminobenzylalcohol, phenol and sodium hydride were purchased from Energy Chemical (Shanghai, China). 1-Methylpyrrolidine and 6-chloropurine were obtained from Bidepharm (Shanghai, China). Hyaluronic acid (HA, MW 5000 Da) was brought from HEOWNS Co., Ltd. (Tianjin, China). N-Hydroxysuccinimide (NHS) and 1-ethyl-3-(3-(dimethylamino) propyl) carbodiimide (EDC) were acquired from Aladdin Reagent Co., Ltd. (Shanghai, China). Reduced nicotinamide adenine dinucleotide phosphate oxidase (NADPH) and coumarin-6 (Cou6) were got from Sigma-Aldrich (St. Louis, United States). BCNU was brought from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Esterase from porcine liver was provided by MACKLIN Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS) was bought from Zhejiang Biological technology stock Co., Ltd. (Zhejiang, China). Rat liver microsomes were obtained from PrimeTox (Wuhan, China). Roswell Park Memorial Institute 1640 Medium (1640) and penicillin–streptomycin solution were purchased from HyClone (Logan City, USA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was provided by Beyotime Biotechnology Co., Ltd (Shanghai, China). Hoechst33342/PI apoptosis detection kit, Annexin V-FITC/PI apoptosis detection kit and Alamar blue were acquired from Solarbio Life Science (Beijing, China). All other reagents were purchased from Beijing Chemicals Co. (Beijing, China) and J&K Scientific Ltd. (Shanghai, China).

Cell culture

The human uterine cervix carcinoma cells (HeLa), the human non-small cell lung cancer cells (A549), the hepatocellular carcinoma cells (SMMC-7721) and mouse brain endothelial cells (b End. 3) were purchased from Peking Union Medical College (Beijing, China). All cells were cultured in RPMI-1640 medium containing 10% FBS, 100 U/mL of penicillin and 100 µg/mL of streptomycin at 37 ℃ using a humidified 5% CO2 incubator (Thermo Fisher Scientific Inc., Waltham, MA, USA). Hypoxic conditions in cell culture were generated by a hypoxia anaerobic incubator chamber (Thermo 1025, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with an appropriate humidified gas mixture containing 1% O2 and 5% CO2 balanced with N2.


BALB/c nude mice (female, 4–6 weeks) were purchased from Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). All animal experiments were conducted under a protocol approved by Beijing University of Technology Institutional Animal Care and Use Committee. Subcutaneous tumor models were planted by injecting 1 × 107 HeLa cells (100 µL) into the left side of each mouse near the armpit. Tumor volume (V) was determined by measuring the length (L) and the width (W), and was calculated according to Eq. (1).

$$\mathrm{V}=\frac{1}{2}L\times {W}^{2}$$


Synthesis of HACB Conjugates

Synthesis of 4-((3-(hydroxymethyl)phenyl) diazenyl) phenol (a)

3-Aminobenzylalcohol (123 mg) was dissolved in 800 µL deionized water. Then, 800 µL hydrochloric acid and 1 mL cold sodium nitrite solution (2 mmol/L) were dropwise added to the above solution stirring for 20 min at 0–4 ℃. After that, 96 µL phenol was added to the solution stirring for 24 h at 0–4 ℃. Then the solution was extracted with distilled water/ethyl acetate (1:1). The organic layers were collected, dehydrated with sodium sulfate and evaporated to get a bronzing solid (0.19 mmol, 19% yield). 1H NMR (400 MHz, DMSO-d6) δ: 10.26 (s, 1H), 7.69–7.83 (m, 4H), 7.43–7.53 (m, 2H), 6.94–6.96 (d, 2H), 5.35–5.38 (t, 1H), 4.60–4.61 (d, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 161.38, 152.56, 145.72, 144.47, 129.48, 128.85, 125.25, 121.69, 119.68, 116.41, 63.00. IR (KBr) υ: 3479.41, 3184.31 (OH), 2881.61 (CH2), 1593 (C=C), 1506.40 (N=N), 1145.05 (C–N), 1145.05 (C–O), 838.95 (=C–H), 694.11 (C–H).

Synthesis of 1-(2-amino-9H-purin-6-yl)-1-methylpyrrolidine-1-chloride (b)

Compound b was synthesized according to the method described by Keppler et al. with modification [37] Briefly, 2-amino-6-chloropurine (2 g) was dissolved in N, N-dimethylformamide (DMF). Then, 1-methylpyrrolidine (3 mL) was added into the DMF solution to react under stirring for 20 h at room temperature. Subsequently, acetone (3 mL) was added to the above solution for precipitation. Then, the precipitates were filtered and washed twice by ether to get a white solid (7.08 mmol, 60% yield). 1H NMR (400 MHz, DMSO-d6) δ: 13.45 (s, 1H), 8.35 (s, 1H), 7.12 (s, 2H), 4.63–4.57 (m, 2H), 4.01–3.95 (m, 2H), 3.66 (s, 3H), 2.34–2.20 (m, 2H), 2.11–2.00 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 161.30, 159.52, 157.63, 150.63, 141.81, 117.07, 65.23, 51.50, 22.37. IR (KBr) υ: 3290.57 (NH2), 3350.92 (NH), 3184.60 (=C–H), 2897.59 (CH2), 1631.80 (C=N), 1564.10 (C=C), 1366.87 (C–C), 1293.28 (C–N), 816.40 (=C–H), 789.91 (N–H), 625.06 (C–H).

Synthesis of 4-((3-(((2-amino-9H-purin-6-yl) oxy) methyl) phenyl) diazenyl) phenol (c)

Compound a (580 mg) was dissolved in 10 mL DMF, to which 600 mg potassium tert-butoxide and 300 mg compound b were added. Then, the mixture was stirred under a nitrogen atmosphere for 6 h at room temperature. Subsequently, 1 mL of deionized water containing 100 µL glacial acetic acid was added to the above solution. Then, the mixture was extracted with ethyl acetate/deionized water (1:1). The organic layers were dried with anhydrous sodium sulfate and concentrated under a vacuum. Finally, the crude product was purified by column chromatography with dichloromethane/methanol (50:1–10:1) as an eluent to obtain a yellow solid (1.28 mmol, 51% yield). 1H NMR (400 MHz, DMSO-d6) δ: 12.48 (s, 1H), 10.37 (s, 1H), 7.93 (s, 1H), 7.84–7.79 (m, 4H), 7.64–7.57(m, 2H), 6.96–6.94 (d, 2H), 6.36 (s, 2H), 5.60 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 161.57, 160.13, 152.67, 145.67, 138.73, 138.41, 130.64, 129.90, 125.39, 122.26, 122.05, 116.43, 66.73. IR (KBr) υ: 3683.45 (OH), 3487.84 (NH2), 3340.02 (NH), 2802.47 (CH), 1587.03 (C=C), 1501.92 (N=N), 1355.59 (C–C), 1283.92 (C–O–C), 1146.54 (C–N), 837.46 (=C–H), 786.69 (N–H), 628.41 (C–H).

Synthesis of HACB conjugates

An aliquot of HA (35 mg, MW =  ~ 5000 Da) was dissolved in distilled water (7 mg/mL) followed by addition of 15 mg EDC and 9 mg NHS, and the mixture was stirred at room temperature for 0.5 h to activate the carboxyl groups of HA. Then, 243 mg of compound c was added to the above solution and stirred at 40 ℃ for 48 h. The obtained crude product was then dialyzed by a dialysis bag (MWCO 3500 Da) against an excess amount of water/DMSO (1:3 to 1:1) for 1 day and against distilled water for another 2 days. The solution was filtered through a microporous membrane with 0.45 µm pores followed by freezing and lyophilizing to obtain a yellowish fluffy powder, named HACB.

Preparation of blank (HACB NPs) or BCNU-loaded micelles (HACB/BCNU NPs)

HACB/BCNU NPs were prepared by solvent volatilization method according to the literature with some modifications [38]. Briefly, a solution of BCNU in ethanol (100 mg/mL) was dropwise added into the aqueous solutions of HACB (25 mg/mL) at different BCNU/HACB ratios (0.5:10, 1:10, 2:10, 3:10, 4:10, w/w) with continuous stirring. For blank micelles, BCNU was omitted. Subsequently, the mixture was sonicated with a probe-type ultrasonicator (Autotune, SONICS Newtown, USA) in an ice bath for 15 min (400 W, working for 5 s, intermittent 2 s). After that, the solution was filtered through a microporous membrane with 0.45 µm pores followed by lyophilizing and stored at − 20 ℃ until further use. For visualization, Cou6-loaded micelles (HACB/Cou6 NPs) were prepared using Cou6 instead of BCNU.

Characterization of HACB NPs and HACB/BCNU NPs

Dynamic light scattering (DLS) measurements were performed to examine the hydrodynamic size, zeta-potential and poly-dispersity (PDI) of HACB NPs and HACB/BCNU NPs using a laser diffraction particle sizer (Nano-ZS, Malvern Instrument, UK). The optical absorption was determined with a UV–vis spectrophotometer (TU-1901, PERSEE, China). The morphology of the micelles was monitored using transmission electron microscopy (TEM, JEM-2100, JEOL, Japan). To investigate the stability of HACB NPs, the mean particle size of the micelles dissolved in H2O, PBS (pH 7.4) or DMEM with 10% FBS was measured at different time points (0, 6, 12, 24, 36 and 48 h).

The encapsulation efficiency (EE) and drug loading (DL) of HACB/BCNU NPs were determined by measuring the concentration of the loaded drugs using HPLC (U3000, Thermo Fisher Scientific, USA) with a C18 column (ZORBAX 5 µm C18, 4.6 × 250 mm, Agilent Technologies Inc., California, USA). Briefly, the total dispersoid was filtered with an Amicon Ultra 10 kD molecular weight centrifugal filter (Millipore, Billerica, MA, USA) to obtain filtrate, which contained the unencapsulated BCNU. Then, the total mixture of the drug was dissolved in ten-fold volume of methanol followed by filtering through 0.22 µm membrane filters. The obtained filtrate was used for the determination of the total BCNU concentration in the solution. All the samples were analyzed by HPLC and the ultraviolet absorption was monitored at 230 nm for BCNU. The values of EE and DL of HACB/BCNU NPs were calculated according to Eqs. (2) and (3):

$$\mathrm{EE}=\frac{Weight \,of \,loaded \,BCNU}{Total \,weight \,of \,BCNU \,in \,the \,reaction \,solution }\times 100$$


$$\mathrm{DL}=\frac{Weight\, of \,loaded \,BCNU}{Total \,weight \,of \,micelles}\times 100$$


Cleavage study of HACB NPs

Hypoxia-sensitivity of HACB NPs

To simulate the hypoxic reductive environment, 1 mM Na2S2O4 was added into the aqueous solution with HACB NPs (1 mg/mL) and the mixture was stirred continuously at 37 ℃. The change of the absorption peak of the azobenzene group in the reaction was recorded by a UV–vis spectrophotometer. Moreover, HACB NPs was incubated with or without Na2S2O4 for 1 h at 37 ℃, and then the hydrodynamic diameter distributions and the morphology were observed by DLS and TEM, respectively.

Esterase responsiveness of HACB NPs

HACB NPs dispersion at a concentration of 1 mg/mL was incubated with or without 30 U/mL porcine liver esterase at 37 ℃ under continuous shaking (100 rpm) for 1 h. After that, the hydrodynamic diameter distributions of the micelles were monitored by DLS, and the morphology was observed by TEM.

In vitro drug release profile

On account of the high instability of BCNU in solution, a Cou6 model was used to examine the in vitro release profile of the micelles. In brief, 2 mL HACB/Cou6 NPs dispersion (containing 2 mg Cou6) was placed into a dialysis bag (MWCO of 1 kDa), and then immersed in 40 mL release media containing 0.5% w/v sodium dodecyl sulfate, porcine liver esterase (0 or 30 U/mL), 790 µg/mL rat liver microsomes and 100 µM NADPH. Air and Nitrogen gas were bubbled through the reaction mixture to create normoxic and hypoxic conditions, respectively. Then, each group was gently shaken at 37 ℃ at a speed of 100 rpm. At the predetermined time point, 0.5 mL of the mixture containing the released Cou6 was drawn for measurement and replaced with fresh medium to maintain a constant volume. Three independent repeats were performed for each sample (n = 3).

Cellular uptake and intracellular release of HACB/Cou6 NPs

Hela cells were seeded in 12-well plates at a density of 1.5 × 105/well and cultured for 24 h at 37 ℃. Then, cells were treated with Cou6 or HACB/Cou6 NPs (Cou6 concentration of 10 ng/mL) for 30 min and 2 h under normoxia and hypoxia. To demonstrate the assisted targeting effect of HA, the cells were pretreated with 5 mg/mL HA for 2 h before exposure to HACB/Cou6 NPs. After treatment, the cells were washed twice with PBS, stained by Hoechst 33342 (10 µg/mL) for 15 min and observed by an IX-51 inverted fluorescence microscope (Olympus Corporation, Tokyo, Japan). To further reveal the intracellular distribution of HACB/Cou6 NPs, the intracellular fluorescence was observed under a confocal laser scanning microscope (CLSM, Nikon AX R, Tokyo, Japan).

In vitro cytotoxicity assay

The cytotoxicity of HACB/BCNU NPs was investigated by an MTT assay. Generally, HeLa cells were seeded in 96-well plates at a density of 5 × 103/well and cultured for 24 h at 37 ℃. Then, the cells were treated with BCNU and HACB/BCNU NPs (at equivalent BCNU concentrations of 10, 20, 50, 100, 200, 400, 600 and 1000 µM) under normoxic and hypoxic conditions. For the combination-treated groups, the cells were pretreated with 40 µM O6-BG for 2 h before exposure to BCNU. Moreover, HeLa cells and a normal cell line b End.3 cells were treated with HACB NPs (0.02, 0.04, 0.10, 0.21, 0.42, 0.83, 1.25 and 2.08 mg/mL) to evaluate the cytotoxicity of blank micelles. After a 24-h treatment, 10 µL MTT solutions (5 mg/mL) were added in each well, and the cells were incubated in the dark for 4 h. Subsequently, the medium was removed and formazan crystals were dissolved in 150 µL DMSO. Finally, the absorbance at 560 nm was determined by a Thermo Scientific Multiskan FC (Multiskan FC, Thermo Scientific, Waltham, MA, USA). The cell viability was calculated according to Eq. (4) as follows:

$$\mathrm{Cell \,viability }\left(\mathrm{\%}\right)={(OD}_{sample}-{OD}_{blank})/( {OD}_{control}-{OD}_{blank})$$


Here, the \({OD}_{sample}\) is the absorbance values of the drug-treated cells, \({OD}_{blank}\) is the absorbance values of blank only containing medium and \({OD}_{control}\) is the absorbance values of the cells without drug treatment.

Live/dead cell staining assay

HeLa cells were seeded in 48-well plates at 8 × 104 cells per well and incubated for 24 h. Afterward, the cells were exposed to BCNU and HACB/BCNU NPs based on BCNU concentrations of 0.1 mM or 0.3 mM under normoxic and hypoxic conditions for 24 h. The combination-treated groups were pretreated with 40 µM O6-BG for 2 h before exposure to BCNU. After that, the cells were stained with 5 µL Hoechst 33,342 and 5 µL PI solution for 15 min. Finally, the cells were washed three times with PBS and observed with a fluorescent microscope (IX-51, Olympus Corporation, Tokyo, Japan).

Colony formation assay

HeLa cells were seeded in 6-well plates at 800 cells per well and maintained for 24 h. Thereafter, the medium was replaced with fresh medium containing BCNU, BCNU + O6-BG or HACB/BCNU NPs (containing 0.05 mM BCNU) under normoxia or hypoxia. The dose of BCNU in each group was 0.05 mM. After treatment for 24 h, the medium was replaced with fresh medium followed by incubation for another 8 days. For the BCNU + O6-BG group, the cells were pretreated by 40 µM O6-BG for 2 h before exposure to BCNU. The cell clones were stained with crystal violet for 10 min followed by washing three times with PBS for observation.

Cell apoptosis

The apoptosis induced by HACB/BCNU NPs was evaluated by Annexin V-FITC/PI apoptosis detection kit. HeLa cells were seeded in 6-well plates at 4 × 105 cells per well and incubated for 24 h. Later, the cells were exposed to BCNU or HACB/BCNU NPs (containing 0.1 mM or 0.3 mM BCNU) under normoxic or hypoxic conditions for 24 h. The combination-treated groups were pretreated by 40 µM O6-BG for 2 h before BCNU exposure. Then, the cells were digested with trypsin without EDTA, centrifuged and washed twice with pre-cooled PBS. The cells were resuspended in a 1 × binding buffer and co-stained with Annexin V-FITC/PI kit for detection by FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA).

Cell wound-healing assay

Wound-healing assay was performed to evaluate the migration of HeLa cells after drug treatment. Briefly, HeLa cells were seeded in 6-well plates at 4 × 105 cells per well and cultured till approximately 90% confluence. Afterward, the medium was removed and a 10 µL pipette tip was used to generate a wound area by scraping the cell monolayer. The cells were treated with BCNU, BCNU + O6-BG or HACB/BCNU NPs under normoxia or hypoxia for 24 h. The concentration of BCNU in each group was 0.1 mM. For the BCNU + O6-BG group, the cells were pretreated with 40 µM O6-BG for 2 h before exposure to BCNU. The wound width was observed and recorded by a fluorescent microscope (IX-51, Olympus Corporation, Tokyo, Japan). The cell migration rate was calculated according to Eq. (5) as follows:

$$\mathrm{Migration\, rate }(\mathrm{\%})=({S}_{0}-{S}_{24})/{S}_{0}\times 100$$


Here, S24 and S0 referred to the area of the region without cells at 24 and 0 h, respectively.

Cell viability in spheroidal cultures

Tumor spheroid culture and morphology analysis

Three-dimensional (3D) tumor spheroids were established to mimic the in vivo hypoxic tumor microenvironment by culturing HeLa cells in a chitosan-hyaluronic acid (C-HA) scaffold, which was prepared according to the method described by Florczyk et al. [39]. HeLa cells were seeded onto the C-HA scaffold in 24-well plates at 5 × 104 cells per scaffold and maintained for 1 h till the cell suspension penetrated the scaffold. Then, 1 mL complete medium was added to each well and the cells were incubated for 10 days at 37 ℃ and 5% CO2 with regular media changes every 2 days. The morphology of the HeLa spheroids inside the C-HA scaffold was acquired on a scanning electron microscope (SEM). The cell-cultured samples were fixed by 2.5% Karnovsky’s fixative overnight at 4 ℃ and dehydrated in a graded series of ethanol (0%, 30%, 50%, 75%, 95%, 100%). After that, the samples were critical-point dried and sputter-coated with gold before imaging with a SU3500 SEM (Hitachi, Tokyo, Japan) [10].

Inhibitory activity of HACB/BCNU NPs against tumor spheroids

HACB/BCNU NPs-induced inhibition of proliferation in tumor spheroids was assessed by Alamar blue assay. After 10 days of incubation, the samples were treated with BCNU, BCNU + O6-BG, HACB/BCNU NPs with proposed concentrations (50, 100, 200, 400 and 1000 µM) in normoxia or hypoxia for 24 h. As described above, cells were exposed to 40 µM O6-BG for 2 h before BCNU exposure for the combination-treated groups. The cells were washed with PBS followed by adding 500 µL medium containing 10% Alamar blue. After incubation for 2 h, 100 µL Alamar blue solution was transferred to a 96-well plate followed by measuring the absorbance at 570 nm and 630 nm. Ultimately, the cell viability (%) was calculated according to Eq. (6) as follows:

$$\mathrm{Cell\, viability }\left(\mathrm{\%}\right)=({E}_{630}\times {OD}_{T570}-{E}_{570}\times {OD}_{T630})/({E}_{630}\times {OD}_{NC570}-{E}_{570}\times {OD}_{NC630})\times 100$$


Here E570 and E630 refer to the absorption coefficient of Alamar blue at 570 nm and 630 nm, respectively. ODT570 and ODT630 are the absorption values of the drug-treated groups at 570 nm and 630 nm, respectively. ODNC570 and ODNC630 are the absorption values of the negative control groups at 570 nm and 630 nm, respectively [10].

In vivo fluorescence imaging

In order to determine the bio-distribution of HACB/BCNU NPs, DiR-labelled HACB NPs were injected into the HeLa tumor xenograft mice via the tail vein followed by observation via a non-invasive near-infrared (NIR) optical imaging technique. When the tumor reached about 300 mm3, the HeLa tumor xenograft mice were randomly divided into three groups and treated with DiR and HACB/DiR NPs both at a dose of 1 mg DiR/kg via the tail vein. In addition, to further confirm the interaction between CD44 receptors and HA, the mice were administrated with a high dose of free HA (1200 mg/kg) via the tail vein 1 h before being treated with HACB/BCNU NPs. At the prearranged time points after injection, NIR fluorescent images were captured by an in vivo imaging system (IVIS, PerkinElmer, United States). Furthermore, major organs (including hearts, livers, spleens, lungs and kidneys) and tumors of each mouse were collected and imaged via the in vivo imaging system.

In vivo synergistic therapeutic efficacy

Tumor growth inhibition of HACB/BCNU NPs was evaluated on HeLa tumor xenograft models. When the tumor reached about 100 mm3, the mice were randomly divided into five groups (five mice per group): (I) control group (PBS); (II) BCNU group at 15 mg BCNU/kg; (III) BCNU + O6-BG group (10 mg/kg of O6-BG was injected intraperitoneally 2 h before BCNU being injected through caudal vein at a dose of 15 mg/kg); (IV) HACB/BCNU NPs-low dose group at 5 mg BCNU/kg; (V) HACB/BCNU NPs-high dose group at 15 mg BCNU/kg. The body weights and the tumor volumes were recorded every 2 days. At day 15, the mice were sacrificed, and the tumors were collected and weighed. All tumors and major organs were fixed in 10% neutral buffered formalin for histological examination. TdT-mediated dUTP nick-end labeling (TUNEL) assay and hematoxylin and eosin (HE) assay were performed to further evaluate the antitumor effect of different formulations. Meanwhile, the major organs were also analyzed by the HE staining at the end of the treatment to determine the biocompatibility and adverse effect of formulations.

Statistical analysis

The data are given as mean ± S. D.. The statistical significance was tested by a two-tailed Student’s t-test or one-way ANOVA. The values between groups were compared using Student’s t-test. A p-value of less than 0.05 was considered statistically significant.