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Targeted delivery of doxorubicin for the treatment of bone metastasis from breast cancer using alendronate-functionalized graphene oxide nanosheets
Journal of Industrial and Engineering Chemistry 76 (2019) 310–317
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Journal of Industrial and Engineering Chemistry journal homepage: www.elsevier.com/locate/jiec
Targeted delivery of doxorubicin for the treatment of bone metastasis from breast cancer using alendronate-functionalized graphene oxide nanosheets Tung Thanh Phama , Hanh Thuy Nguyena , Cao Dai Phunga , Shiva Pathaka , Shobha Regmia , Dong-Ho Haa , Jong Oh Kima , Chul Soon Yonga , Sang Kyoon Kimb , Ji-Eun Choic , Simmyung Yookd,* , Jun-Beom Parke,* , Jee-Heon Jeonga,* a
College of Pharmacy, Yeungnam University, Gyeongsan, Gyeongbuk 38541, South Korea Laboratory Animal Center, Daegu–Gyeongbuk Medical Innovation Foundation (DGMIF), Daegu 41061, South Korea c Department of Clinical Medicinal Sciences, PRIME College of Interdisciplinary & Creative Studies, Konyang University, Nonsan, Chungnam 32992, South Korea d College of Pharmacy, Keimyung University, Daegu 42601, South Korea e Department of Periodontics, Seoul St Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 November 2018 Received in revised form 8 March 2019 Accepted 29 March 2019 Available online 8 April 2019
Selective delivery of anti-cancer drugs to bone tumors remains an on-going developmental issue due to problems of drug availability and the physiological nature of bone. This study was undertaken to enhance accumulation of doxorubicin (DOX) in bone metastasis microenvironments using alendronatefunctionalized graphene oxide nanosheets (NGO-ALs). In vivo biodistribution study showed NGO-ALs were retained for longer and at higher concentrations in bone tumor areas than non-functionalized NGOs. Our findings suggest that NGO-ALs could be used as a promising carrier to enhance antitumor effects and diminish the off-target effects of DOX for the treatment of bone metastasis. © 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Keywords: Bone metastasis Graphene oxides Alendronate Targeted delivery
Introduction The skeletal system is the most frequent target of breast cancer metastasis [1,2]. Approximately 70% of patients with advanced breast cancer experience bone metastases [3], which can lead to incurable osteolytic and osteoblastic lesions, and result in so-called skeletal-related events (SREs), that are, pathologic fractures, hypercalcemia, spinal cord compression, requiring surgical intervention and palliative radiotherapy. Furthermore, SREs are associated with reduced survival, poor quality of life, and substantial medical care costs [4,5]. Conventional chemotherapies for bone metastasis are limited in terms of their severe side effects and their poor anti-tumor efficacies due to the hardnesses, poor permeabilities, and physiological and biochemical processes of bone. Therefore, it is essential that selective-delivery methods be devised to enable chemotherapeutics to target bone microenvironments and to minimize their side effects.
* Corresponding authors. E-mail addresses: [email protected] (S. Yook), [email protected] (J.-B. Park), [email protected] (J.-H. Jeong).
Human bone is a heterogeneous composite comprised of an inorganic phase (60%; hydroxyapatite), an organic phase (30%; bone matrix protein) and water (10%) [6]. The unique characteristics of the highly calcified skeletal microenvironment have been widely utilized to develop bone-homing therapy. Poly-amino acids [7–10], tetracycline [11–13] and bisphosphonates (BPs) [14–17] have been reported to be potential bone-targeting ligands due to their remarkably strong affinities for hydroxyapatite (HA). Among these targeting molecules, BP-based ligands have attracted considerable attention because they alleviate bone pain, reduce risks of osteoporotic complications, and are well-tolerated and safe [18]. The accumulation of BPs in metastatic bone lesion has been reported to be 10- to 20-fold higher than that in healthy bone [19– 21]. In the present study, alendronate (AL), a second-generation bisphosphonate approved by FDA to treat tumor-associated hypercalcemia and several bone-related diseases, was conjugated to graphene oxide nanosheets (NGOs) to facilitate the targeted delivery of doxorubicin (DOX) to bone metastases (Fig. 1). Strong chelation between AL and HA was expected to enhance accumulation of NGOs in the skeletal system and reduce accumulations in other organs. In addition, the two-dimensional structures and extremely high surface areas of NGOs have been reported to enable
https://doi.org/10.1016/j.jiec.2019.03.055 1226-086X/© 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
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Synthesis of NGO-ALs AL was covalently conjugated to NGOs using the cross-linkers EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide ester). Briefly, 5.75 mg of NHS, 8.85 mL of EDC (both from Tokyo Chemical Industry Co., Ltd., Chuo-ku, Tokyo) and 10 mL of NGO suspension (0.5 mg/mL) were placed in sealed glass vial, sonicated for 2 h using a bath sonicator, and stirred for 4 h at room temperature. Then, co-products and excess reactants were removed from the suspension by washing 5 times with bicarbonate buffer (pH 8.5; 0.01 M). Alendronate trihydrate (16.26 mg; Sigma Aldrich, St. Louis, MO) was added to 10 mL of the suspension obtained, sonicated for 30 min and stirred overnight. Finally, NGO-ALs were purified by centrifuging at 27,230 g and reconstituting with distilled water ten times. Characterization of NGO-ALs Physiochemical characterizations The morphologies and sizes of NGO-ALs were determined by transmission electron microscopy (TEM). Briefly, a drop of an NGOAL suspension (0.01 mg/mL) was placed on a carbon-coated copper grid, dried at room temperature, and observed under a transmission electron microscope (H7600; Hitachi, Tokyo) at an accelerating voltage of 100 kV. FT-IR (Fourier transform-infrared) spectra of formulations were obtained using a Thermo Scientific Nicolet Nexus 670 FT-IT Spectrometer and Smart iTR (Thermo Fisher Scientific Inc., Waltham, MA) equipped with a diamond window.
Fig. 1. Bone-targeted delivery of DOX for treating bone metastasis from breast cancer. (A) Schematic illustration showing the construction of DOX@PEG-NGO-AL complexes. (B) Targeted delivery of DOX to bone tumor microenvironment by strong chelation of AL with hydroxyapatite and pH-responsive release of DOX from DOX@PEG-NGO-ALs.
Phosphate assay Amounts of AL that conjugated with NGOs were determined using a phosphate assay [26]. Briefly, 50 mL of an NGO-AL suspension (1 mg/mL), 3 mg of magnesium nitrate and 50 mL ethanol was added in a glass tube and heated over a strong flame until brown fumes disappeared. The ash was then treated with 0.3 mL hydrochloride acid (0.5 N; Sigma Aldrich, St. Louis, MO) at 100 C for 15 min, and then with 0.6 mL ammonium molybdate tetrahydrate (0.42% w/v in 1 N H2SO4; Sigma Aldrich, St. Louis, MO) and 0.1 mL ascorbic acid (10%, w/v; Sigma Aldrich, St. Louis, MO) at 37 C for 15 min. Solution absorbances were measured at 820 nm, and AL concentrations were read off standard calibration curves. NGO suspension was used as the negative control. Loading of DOX onto NGO-ALs
DOX loadings greater than those possible using other carriers [22]. Furthermore, DOX release from NGOs has been showed to be pHdependent [22–24], which suggests drug release would be accelerated in the acidic microenvironments of bone tumors and those created by resorption, and that DOX@PEG-NGO-AL nanocomposites offer promise for the treatment of bone metastasis. This study was undertaken to determine whether PEG-NGO-ALs enhance DOX accumulation in metastatic bone tumors in advanced breast cancer. Materials and methods
To incorporate DOX with NGO-ALs, 10 mL of doxorubicin hydrochloride solution (0.5 mg/mL) was added to 10 mL of NGOAL suspension (1 mg/mL). The mixture was then sonicated for 1 h and stirred overnight at room temperature. Free DOX was removed by repeated centrifugation at 5640 g using an ultracentrifugal filter tube (MWCO: 300 kDa; Vivaproducts Incs, Littleton, MA). Drug loading efficiencies (LEs) and drug loading capacities (LCs) were indirectly determined by measuring the concentration of free DOX in the filtrates. The concentration of DOX was quantified by UV–Vis absorbances at 481 nm using a microplate reader (Spark 10M; Tecan Australia, Port Melbourne, VIC, Australia).
Synthesis of NGOs Grafting of DSPE-PEG onto NGO-ALs Graphene oxide sheets (GOs) were prepared by modified Hummer’s methods using flake expandable graphite (Alfa Aesar, Thermo Fisher Scientific, Waltham, MA), as previously described [25]. Then, 10 mL of GO suspension (1 mg/mL) was then sonicated at 700 W for 2 h using a probe sonicator (VCX 750; Sonic1, Sonics & Materials, Inc, Newtown, CT) to produce exfoliated NGOs. Large GO sheets in suspension were further removed by centrifugation at 9425 g for 10 min.
To increase the stability of NGO derivatives under physiological conditions, DSPE-PEG (MW: 5000 Da; Nanocs, Boston, MA) was grafted onto NGOs, DOX@NGOs, NGO-ALs, and DOX@NGO-ALs by simply mixing DSPE-PEG with NGO-ALs in distilled water for 6 h. The stabilities of NGO-AL formulations with and without DSPEPEG were evaluated in phosphate buffer saline (PBS; 1X), fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT), or DMEM
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containing 10% FBS. In brief, formulations were added to media to a final concentration of 0.1 mg/mL and incubated at 37 C. After 24 h of incubation, the appearance of aggregation in each sample was assessed. The detachment of DSPE-PEG from nanosheets was investigated by using Cy5.5-labeled DSPE-PEG (Cy5.5-DSPE-PEG; MW: 5000 Da; Biochempeg, Watertown, MA). Briefly, DSPE-PEGgrafted formulations suspended in PBS, FBS, or DMEM containing 10% FBS were incubated at 37 C for 48 h. At predetermined times, samples were taken and centrifuged at 27,230 g for 1 h. The fluorescence intensities of free Cy5.5-DSPE-PEG in media were then measured using a microplate reader (Spark 10M; Tecan Australia; Port Melbourne, VIC, Australia). In vitro release study The release of DOX from DOX@PEG-NGO-ALs was assessed in phosphate buffer solution (PBS; pH 7.4) and acetate buffer solution (ABS; pH 4.5) by dialysis. A DOX weight equivalent of 0.1 mg of DOX@PEG-NGO-ALs was placed in a sealed dialysis bag (MWCO: 3500 Da; Spectra/Por, Spectrum Laboratories Inc., Rancho Dominguez, CA). The bag was then placed in a 50-mL conical tube containing 20 mL of either PBS or ABS. At regular time intervals, samples were withdrawn for analysis. Volume replenishment was performed using equal volumes of fresh medium. The concentrations of DOX in the release media were determined by UV–Vis spectrophotometry as described above. In vitro hydroxyapatite binding assay Hydroxyapatite (HA) microparticles (Sigma Aldrich, St. Louis, MO) were used to evaluate the affinity of NGO-ALs for bone. For this purpose, HA microparticles (20 mg) were mixed with 1 mL of either NGOs or NGO-ALs (0.1 mg/mL) in microtubes, and shaken for 4 h. At predetermined intervals (0.5 h, 1 h, 2 h, and 4 h), HA microparticles were allowed to settle, and supernatants were withdrawn for analysis. The UV absorbances of suspensions at 234 nm before (Abbefore) and after (Abafter) mixing with HA recorded using a UV–Vis spectrophotometer (Perkin Elmer U2800; Hitachi, Tokyo, Japan) were used to calculate the concentration of nanosheets. Weights of nanosheets bound to HA microparticles (Wb) were calculated using the following formula. Wb ðmgÞ ¼
Cbefore Cafter 100 Cbefore
Cbefore refers to the concentration of samples before mixing with HA powder, Cafter refers to the concentration of samples after mixing with HA powder. Similarly, decreases in fluorescence intensity at excitation and emission wavelengths of 673 nm and 797 nm, respectively, were used to examine the affinity of Cy5.5-labeled PEG-NGO-ALs for HA. Furthermore, HA microparticles in the pellets were washed 5 times with distilled water, freeze-dried, and observed under a scanning electron microscope (S-4100; Hitachi, Tokyo, Japan). Cell culture and growth condition MDA-MB-231 cells (a human breast carcinoma cell-line) were obtained from the Korean Cell Line Bank (Jongro-gu, Seoul, South Korea). Cells were cultured in DMEM containing 0.45% (w/v) glucose, 10% (v/v) FBS (Hyclone Laboratories, Logan, UT) and 1% (v/ v) penicillin-streptomycin (Hyclone Laboratories, Logan, UT). Cell viability assay The cytotoxic effects of NGOs, free DOX, PEG-NGOs, PEG-NGOALs, DOX@PEG-NGOs, and, DOX@PEG-NGO-ALs on MDA-MB-231 cells were evaluated using a cell counting kit-8 (CCK-8; Dojindo
Molecular Technology Inc., Rockville, MD) assay. Briefly, cells were cultured in a 96-well plate (Corning, Steuben County, NY) at a density of 5 103 cells/well. Samples were added to wells; the same volumes of PBS were added as negative controls. Following treatment for 24 h, cells were washed twice with PBS. The medium in each well was then replaced by an equivalent volume of fresh medium. WST-8 [10 mL; 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoium, monosodium salt] solution was then added to each well, and the plate was placed in a cell incubator for 3 h. After that, 70 mL of medium in each well was transferred into another 96-well plate for absorbance measurement at 450 nm using a plate reader (Spark 10M; Tecan Australia, Port Melbourne, VIC, Australia). Relative cell viabilities were calculated using the following formula. Cell viability ð% of controlÞ ¼
Absample Abblank 100% Abcontrol Abblank
Absample refers to the groups of cells treated with samples. Abcontrol refers to the groups treated with PBS. Abblank refers to medium only. Effect of DOX@PEG-NGO-ALs on an in vitro bone metastasis model An in vitro model, in which MDA-MB-231 cells were cultured in a three-dimensional collagen gel containing HA particles, was used to assess the effect of DOX@PEG-NGO-ALs on bone metastasis from breast cancer. Briefly, a bovine collagen solution (2.305 mL; 3 mg/mL; PureCol1, Advanced BioMatrix, CA) was mixed with a 20X PBS solution (0.188 mL), a sodium hydroxide solution (0.32 mL; 0.1 M), and distilled water (0.188 mL) at 4 C. Next, 10 mg of HA particles and 5 103 MDA-MB-231 cells were gently suspended in 100 mL of obtained collagen solution. The suspension was then transferred to a 96-well plate, which was pre-incubated at 40 C for 2 h. The plate was placed in a cell incubator for 3 h for complete gelation. The gel was washed twice with PBS and flipped using a sterile pipette tip to expose the HA surface. To evaluate the anti-tumor effect of DOX@PEG-NGOs and DOX@PEG-NGO-ALs, 100 mL of DOX@PEGNGOs and DOX@PEG-NGO-ALs suspension (equivalent to 25 mg DOX/mL) was added to the gels and incubated for 30 min under slight agitation. Same volumes of PBS were added as negative controls. The supernatant in each well was then removed and the gels were washed 3 times with PBS. The gels were incubated in 100 mL of culture media for 24 h. Finally, the cell viability was assessed using CCK-8 assay as described in Section “Cell viability assay”. In vivo biodistribution study The in vivo biodistribution of PEG-NGOs and PEG-NGO-ALs were investigated in immunocompromised BALC/c nude mice (Male; Orient-Bio Inc., Joongwon, Seongnam, South Korea). PEGNGOs and PEG-NGO-ALs were fluorescently labeled using DSPEPEG-Cy5.5. Sample stabilities in physiological salt buffer and affinities for HA were confirmed before administration. For wholebody imaging, 100 mL of a suspension of either PEG-NGOs or PEGNGO-ALs (2 mg/mL) was injected intravenously, and the distribution of fluorescence was recorded at day 1 and day 3 after injection using an in vivo imaging system (IVIS). The accumulation of nanosheets in the skeletal system was further assessed using an IVIS SpectrumCT in vivo imaging system (Caliper Lifescience, Hopkinton, MA). The distribution of fluorescence was analyzed based on a reconstructed three-dimensional diffuse tomography using Live Image1 3.1 software. To evaluate the selective delivery of nanosheets to bone tumors, an animal model of breast cancer bone metastasis was developed by injecting human breast adenocarcinoma MDA-MB-231 cells into the tibias of BABL/c nude mice, as previously reported [27].
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Briefly, mice were anesthetized with ketamine (80 mg/kg; Huons, Gyeonggi, South Korea) and xylazine (16 mg/kg; Bayer Korea Ltd., Seoul, South Korea), and a 31-gauge needle was then pushed through the patellar ligament into the tibia. A cell suspension (10 mL; 107 cells/mL) was then slowly injected into the marrow cavity. After 5 weeks, mice with a bone tumor were selected for in vivo distribution study and injected with Cy5.5-labeled nanosheets (100 mL; 2 mg/mL) via a tail vein. Whole body and tibia fluorescence intensities were recorded after 24 h using a fluorescently-labeled organism bioimaging instrument (FOBI; Neoscience, Suwon, Gyeonggi, South Korea). Statistical analysis Data were expressed as the mean standard deviation. The unpaired t-test or one-way ANOVA test was performed for the statistical analyses. P values < 0.05 were regarded as statistically significant. Results and discussion Characterization of NGOs GO flakes were prepared by chemically-induced oxidation of graphite using modified Hummer’s method [28]. FT-IR analysis confirmed the formation of new functional groups in GOs (Fig. S1A). A broad and intense peak at 3400 cm1 was ascribed to hydroxyl groups, a peak at approximately 1730 cm1 to the stretch of C¼O carboxylic moieties, and a peak at 1620 cm1 to aromatic C–C bonds. Peaks at 1365, 1215 and 1054 cm1 were ascribed to C–O–H deformation, C–H stretch (epoxy groups), and C–O stretch vibrations (alkoxy groups), respectively. NGOs were prepared by ultrasonication as previously described [29]; sonication time was optimized beforehand. After 2 h of sonication, mean dynamic particle sizes of GO sheets reduced
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significantly from approximately 750 nm to <200 nm (Fig. S1B). TEM revealed thin nanosheets with maximum dimensions of 60– 150 nm (Fig. 2A). FT-IR analysis confirmed functional groups of NGOs were not significantly altered by ultrasonication (Fig. S1B). UV–Vis spectrophotometry exhibited the characteristic peaks of NGOs, that are, a peak at 234 nm due to p–p* transition of aromatic C–C bonds and a shoulder at 300 nm due to n–p* transitions of C¼O bonds. Characterization of NGO-ALs NGOs were functionalized with AL due to covalent bond formation between the amine group of AL and the carboxylic acid groups of NGOs using EDC/NHS as cross-linkers. The FT–IR analysis revealed that the intensity of the peak at 1730 cm1 (corresponding to the carboxyl groups) of NGOs was significantly diminished, and that a new prominent peak related to stretching mode of amide groups appeared at 1631 cm1 (Fig. 2C), suggesting the formation of amide linkages between the carboxylic acid groups of NGOs and the amine group of AL. Additionally, two small peaks at 1020 cm1 and 926 cm1 appeared, which correspond to hydroxyl (–OH) group bending and P¼O stretching vibrations of AL. The amount of AL in NGO-ALs was 0.55 0.02 mg per 1 mg as determined in triplicated using phosphate assay. Loading and pH-triggered release of DOX DOX was non-covalently loaded onto NGO-ALs by simply mixing DOX and NGO-AL suspension with slight sonication. The formation of DOX@NGO-ALs was confirmed by UV–Vis spectrophotometry. As indicated in Fig. 2B, after removing free DOX, the characteristic absorption peaks of DOX at 233 nm and 480 nm were observed in the UV–Vis spectra of NGO-ALs, suggesting the stable incorporation of DOX. Notably, the DOX loading efficiency (LE) and loading capacity (LC) of DOX@NGO-ALs were 98.51 0.45% and 99.09 0.26%, respectively.
Fig. 2. Physiochemical characterization of DOX@PEG-NGO-ALs. (A) Transmission electron microcopy image of NGO-ALs. Scale bar: 100 nm. (B) UV–Vis spectra and optical image of DOX (red), NGO-ALs (blue) and DOX@NGO-ALs (green). (C) Fourier transform-infra red spectra of AL, NGO-ALs, DSPE-PEG, PEG-NGO-ALs, DOX, DOX@PEG-NGO-ALs. (D) Release profiles of DOX from DOX@PEG-NGO-ALs in acetate buffer (ABS; pH 4.5) and phosphate buffer (PBS; pH 7.4). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Szabó et al. reported that NGOs have extremely high surface areas (2600–2800 m2/g) [30], and thus, provide large surfaces for drug adsorption on edges and side of single atom thick two-dimensional NGO structures. This unique characteristic allows NGOs to possess remarkably higher LCs than other nano-carriers [31]. The high LE observed for DOX@NGO-ALs could be explained by strong p–p stacking interactions between the sp2-hybridized p bonds of NGO-ALs and the quinine portion of DOX and by hydrogen bonding between the carboxylic acid (–COOH), hydroxyl (–OH) groups of NGOs and the amine (–NH2), hydroxyl (–OH) groups of DOX. Interestingly, the release of DOX from DOX@NGO-ALs occurred in a pH-dependent manner. As indicated in Fig. 2D, only 16.87 2.84% of DOX was released after 60 h under neutral conditions (pH 7.4), whereas more than 60% of DOX was released at pH 4.5. This accelerated release under acidic conditions could be due to the partial protonation of the hydroxyl and amine groups of DOX, leading to higher drug solubility and weakening of hydrogen bonds between DOX and NGOs [30]. This pH stimuli-responsive behavior plays an important role in targeting effect of DOX@NGO-ALs since the acid–base balance in skeletal tissue is lost when bone metastasis occurs [32,33]. The predominant activity of osteoclasts in the tumor microenvironment increases the
concentration of H+ and acidic hydrolases in the bone resorption compartment and results in the digestion of the mineral and organic phases of bone matrix. Subsequently, pH values in osteolytic lesions formed by bone tumors decrease to approximately 4.5 [34]. Subsequenly, it is possible that slow release under neutral conditions and rapid release under acidic conditions may reduce the toxic effect of DOX on normal tissue and enhance anti-tumor efficiency. In vitro bone targeting The HA binding assay was conducted to evaluate the affinity of NGO-ALs for bone. HA microparticles were used as an in vitro model of bone because, as mentioned above, HA is the major mineral component of bone. As shown in Fig. 3B, NGO-ALs exhibited significantly higher affinity for HA than NGOs. After 1 h of mixing, more than 50% of NGO-ALs bound to HA microparticles, and this percentage gradually increased to approximately 70% at 4 h. In contrast, only 10.67% of NGOs bound to HA even after 4 h of mixing. In addition, the color of HA powder changed to grey and red after incubation with NGO-ALs and DOX@NGO-ALs, respectively, whereas no such change was observed for NGOs (Fig. 3C).
Fig. 3. In vitro hydroxyapatite binding assay. (A) Methodology to evaluate the affinity of NGOs and NGO-ALs to hydroxyapatite. (B) Affinity of NGOs and NGO-ALs to hydroxyapatite. (C) Digital image of hydroxyapatite powder after 4 h of mixing with NGOs, NGO-ALs and DOX@NGO-ALs. (D) SEM image of HA microparticles before and after 4 h of mixing with NGOs and NGO-ALs. Scale bar: 1 mm.
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Further assessment of the surface morphology of HA by SEM revealed dense accumulations of NGO-ALs on the surface of microparticles, while NGOs were barely observed on the surfaces of microparticles after mixing (Fig. 3D). These results indicate that NGO-ALs have a remarkably strong affinity for HA, suggesting that AL-functionalized NGOs offer a means of delivering drugs to bone. Incorporation of DSPE-PEG onto DOX@NGO-ALs NGOs aggregate in the presence of salts in physiological buffers due to the charge screening effect. To address this problem, researchers have surface-modified NGOs with hydrophilic polymers to improve their stabilities [35–38]. Similarly, in this study, PEG was used as the stabilizer due to its solubility and biocompatibility. DSPE-PEG was grafted hydrophobically onto NGO-ALs using the interaction between the lipid chain of DSPE and sp2-hybridized p-conjugated structure of NGOs. DSPE-PEG was added to nanosheet suspension of NGO-ALs at a weight ratio of 1:1 without any significant change in affinity for HA (Fig. S3A–B). Both
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PEG-NGO-AL and DOX@PEG-NGO-AL suspensions proved stable in PBS and DMEM containing 10% FBS for 24 h, whereas in the absence of DSPE-PEG, the agglomeration was clearly observed (Fig. S3C). In addition, we examined DSPE-PEG loss from these NGOs in three different media: PBS (pH 7.4), FBS, and DMEM containing 10% FBS. Less than 10% of Cy5.5-DSPE-PEG was found to be released after 48 h of incubation in all three media (Fig. S3D). These results suggest that DSPE-PEG might be an effective stabilizer for preventing DOX@NGO-AL aggregation under physiological conditions. Furthermore, the incorporation of Cy5.5-DSPE-PEG onto NGO-ALs provides a means for fluorescent-labeling nanosheets. In vitro cytotoxicity Cytotoxicities of DOX, PEG-NGO-ALs and DOX@PEG-NGO-ALs on MDA-MB-231 cell line were investigated using a CCK-8 assay. PEG-NGOs and PEG-NGO-ALs had no obvious toxic effect even at 100 mg/mL (Fig. 4A). In addition, DOX and DOX@PEG-NGO-ALs exhibited similar time-dependent toxic effects on MDA-MB-231
Fig. 4. In vitro anti-tumor effect of DOX@PEG-NGO-ALs. (A) Cytotoxicity of PEG-NGOs and PEG-NGO-ALs at concentration of 1.25 mg/mL, 6.25 mg/mL, 12.5 mg/mL, 25 mg/mL, 50 mg/mL, 100 mg/mL. (B) Concentration-dependent cytotoxicity of free DOX and DOX@PEG-NGO-ALs. (C) Anti-tumor effect of DOX@PEG-NGOs and DOX@PEG-NGO-ALs assessed using a co-culture model of MDA-MB-231 cells and hydroxyapatite particles in three-dimensional collagen gel.
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cell line (Fig. 4B), suggesting the incorporation of DOX with PEGNGO-ALs did not reduce its therapeutic potency. To further assess the potential of DOX@PEG-NGO-ALs for treating bone metastasis from breast cancer, we developed an in vitro bone tumor model, in which MDA-MB-231 cells were co-cultured with HA particles in a three-dimensional collagen gel (Fig. 4C). Since the physiological structure of bone mainly consists of HA and bone matrix proteins, collagen gel containing HA particles can relatively imitate the natural environment of bone for the growth of breast cancer cells. Interestingly, DOX@PEG-NGO-ALs exhibited significantly higher toxicity as compared to DOX@PEG-NGOs on MDA-MB-231 cells in our in vitro bone cancer model. This suggests the superiority of DOX@PEG-NGO-ALs for treating bone tumor as compared to nonfunctionalized DOX@PEG-NGOs. In vivo bone-homing effects To investigate the role of the AL component on the bonehoming ability of PEG-NGO-ALs, Cy5.5-labeled PEG-NGOs and Cy5.5-labeled PEG-NGO-ALs were injected into BALB/c nude mice via a tail vein. IVIS images revealed that PEG-NGO-ALs were
Fig. 5. In vivo biodistribution of PEG-NGOs and PEG-NGO-ALs on day 3 after intravenous injection.
retained in mice body longer than PEG-NGOs (Figs. 5A and S4), which concurs with previous studies [17,39]. In addition, PEGNGOs mostly accumulated in liver and spleen, whereas PEG-NGOALs were observed liver, spleen, femurs, and tibias. Similarly, Dhifaf et al. described that 24 h after injection, PEG-NGOs accumulated in organs of the reticuloendothelial system (RES), including liver and spleen, and eventually be eliminated by urinary excretion [40]. In another study, the blood circulation half-life of NGOs and PEG-NGOs were reported to be 5.35 h and 6.29 h, respectively [41]. After entering the blood stream, NGOs may tend to roll and fold to form smaller particles, and thus, are able to cross the glomerular filtration barrier [40,42,43]. On the other hand, larger NGOs may be uptaken by the RES and localize in liver and spleen [40,44]. This could be why PEG-NGOs accumulated in RES organs and fluorescence intensity rapidly diminished on days 1 and 3 post-injection in the PEG-NGOs treated mice. We speculate that the greater retention time of PEGNGO-ALs as compared with non-functionalized NGOs could be due to interaction with the skeletal system, and the confinement of nanosheets to bone might delay entrapment by the RES and accessibility to the glomerular filtration barrier, and thus, increase in vivo retention. The accumulation of nanosheets in the skeletal system on day 3 post-injection was examined by IVIS/ micro CT imaging (Fig. 5B). NGO-ALs were clearly observed in femurs, tibias, and ribs, whereas NGO fluorescence was barely found in the skeletal system. These results suggest that functionalization of NGOs with AL increases NGO accumulation in the skeletal system and enhances in vivo retention times after intravenous administration.
Fig. 6. The preferential localization of PEG-NGO-ALs at the site of bone tumor. (A) Optical image and fluorescence image of healthy tibiae and tibiae bearing tumor after 24 h post-injection of Cy5.5-labeled PEG-NGO-AL. (B) Quantitative measurement of fluorescence intensity in healthy tibiae and tibiae bearing tumor after 24 h post-injection of Cy5.5-labeled PEG-NGO-AL (n = 4). **p < 0.05.
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The different biodistributions of PEG-NGO-ALs in healthy bones and bone bearing tumors were also evaluated using a murine bone cancer model, in which the intra-tibial inoculation of MDA-MB231 cells was used to mimick the final stage of bone metastasis in breast cancer. Though the early metastatic steps are bypassed, this model provides similar tumor-takes and osteolytic lesion development rates among recipients [27]. In addition, this model enables the role played by the bone tumor microenvironment to be evaluated without concerns about interference by tumors in other sites. We assessed the fluorescence intensities of heathy tibias isolated from controls and tumor-bearing tibias 24 h after injection. As shown in Fig. 6, fluorescence intensities in tibiabearing tumors were significantly higher as compared to fluorescence intensities in the healthy tibia (p < 0.05; n = 4), indicating the preferential accumulation of nanosheets in bone cancer microenvironments. This phenomenon might be due to greater HA exposure in such regions. As previously reported, bone surfaces are generally covered by cells and organic matrix, which might reduce AL binding, whereas bone remodeling during osteolytic lesion development leads to the digestion of bone organic matrix and cell detachment [45,46], which would provide a more accessible surface for AL chelation. This selective accumulation suggests that PEG-NGO-ALs are viewed as a potential carrier for the targeted delivery of drugs to sites of bone metastasis. Conclusion In summary, this study describes a promising strategy for treating bone metastasis in advanced breast cancer using DOX@PEG-NGO-ALs. The DOX@PEG-NGO-AL formulation exhibited rapid DOX release under acidic condition which mimics the microenvironment of bone tumors. Most importantly, AL functionalization significantly increased the retention and accumulation of nanosheets in the murine skeletal system, especially in osteolytic lesions. These findings suggest PEG-NGO-ALs provide a potential strategy for enhancing antitumor effects and for diminishing the off-target toxicities of anticancer drugs used for the treatment of bone cancer. The unique ability of NGOs to carry and preserve the stabilities of different therapeutic agents, which include growth factors (BMP-2, VEGF), siRNAs, and plasmid DNAs, encourages us to believe that application of the PEG-NGO-ALs platform could be extended to the treatments of other bonerelated diseases, such as, osteoporosis and Paget’s disease. Declaration of interest The authors have no conflict of interest to declare. Acknowledgments This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Korean Ministry of Science, ICT, and Future Planning (grant no. 2015R1A5A2009124) and funded by the Ministry of Education (grant no. 2017R1D1A1B03027831); and by the Korea Health Technology R & D Project through the Korea Health Industry Development Institute (KHIDI) and the Korean Ministry of Health and Welfare (grant no. HI16C1767 and HI18C045 3); and by the Creative Economy Leading Technology Development Program through the Gyeongsangbuk-Do and Gyeongbuk Science and Technology Promotion Center of Korea (grant no. SF316001A).
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Targeted delivery of doxorubicin for the treatment of bone metastasis from breast cancer using alendronate-functionalized graphene oxide nanosheets Tung Thanh Phama , Hanh Thuy Nguyena , Cao Dai Phunga , Shiva Pathaka , Shobha Regmia , Dong-Ho Haa , Jong Oh Kima , Chul Soon Yonga , Sang Kyoon Kimb , Ji-Eun Choic , Simmyung Yookd,* , Jun-Beom Parke,* , Jee-Heon Jeonga,* a
College of Pharmacy, Yeungnam University, Gyeongsan, Gyeongbuk 38541, South Korea Laboratory Animal Center, Daegu–Gyeongbuk Medical Innovation Foundation (DGMIF), Daegu 41061, South Korea c Department of Clinical Medicinal Sciences, PRIME College of Interdisciplinary & Creative Studies, Konyang University, Nonsan, Chungnam 32992, South Korea d College of Pharmacy, Keimyung University, Daegu 42601, South Korea e Department of Periodontics, Seoul St Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 27 November 2018 Received in revised form 8 March 2019 Accepted 29 March 2019 Available online 8 April 2019
Selective delivery of anti-cancer drugs to bone tumors remains an on-going developmental issue due to problems of drug availability and the physiological nature of bone. This study was undertaken to enhance accumulation of doxorubicin (DOX) in bone metastasis microenvironments using alendronatefunctionalized graphene oxide nanosheets (NGO-ALs). In vivo biodistribution study showed NGO-ALs were retained for longer and at higher concentrations in bone tumor areas than non-functionalized NGOs. Our findings suggest that NGO-ALs could be used as a promising carrier to enhance antitumor effects and diminish the off-target effects of DOX for the treatment of bone metastasis. © 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Keywords: Bone metastasis Graphene oxides Alendronate Targeted delivery
Introduction The skeletal system is the most frequent target of breast cancer metastasis [1,2]. Approximately 70% of patients with advanced breast cancer experience bone metastases [3], which can lead to incurable osteolytic and osteoblastic lesions, and result in so-called skeletal-related events (SREs), that are, pathologic fractures, hypercalcemia, spinal cord compression, requiring surgical intervention and palliative radiotherapy. Furthermore, SREs are associated with reduced survival, poor quality of life, and substantial medical care costs [4,5]. Conventional chemotherapies for bone metastasis are limited in terms of their severe side effects and their poor anti-tumor efficacies due to the hardnesses, poor permeabilities, and physiological and biochemical processes of bone. Therefore, it is essential that selective-delivery methods be devised to enable chemotherapeutics to target bone microenvironments and to minimize their side effects.
* Corresponding authors. E-mail addresses: [email protected] (S. Yook), [email protected] (J.-B. Park), [email protected] (J.-H. Jeong).
Human bone is a heterogeneous composite comprised of an inorganic phase (60%; hydroxyapatite), an organic phase (30%; bone matrix protein) and water (10%) [6]. The unique characteristics of the highly calcified skeletal microenvironment have been widely utilized to develop bone-homing therapy. Poly-amino acids [7–10], tetracycline [11–13] and bisphosphonates (BPs) [14–17] have been reported to be potential bone-targeting ligands due to their remarkably strong affinities for hydroxyapatite (HA). Among these targeting molecules, BP-based ligands have attracted considerable attention because they alleviate bone pain, reduce risks of osteoporotic complications, and are well-tolerated and safe [18]. The accumulation of BPs in metastatic bone lesion has been reported to be 10- to 20-fold higher than that in healthy bone [19– 21]. In the present study, alendronate (AL), a second-generation bisphosphonate approved by FDA to treat tumor-associated hypercalcemia and several bone-related diseases, was conjugated to graphene oxide nanosheets (NGOs) to facilitate the targeted delivery of doxorubicin (DOX) to bone metastases (Fig. 1). Strong chelation between AL and HA was expected to enhance accumulation of NGOs in the skeletal system and reduce accumulations in other organs. In addition, the two-dimensional structures and extremely high surface areas of NGOs have been reported to enable
https://doi.org/10.1016/j.jiec.2019.03.055 1226-086X/© 2019 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
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Synthesis of NGO-ALs AL was covalently conjugated to NGOs using the cross-linkers EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide ester). Briefly, 5.75 mg of NHS, 8.85 mL of EDC (both from Tokyo Chemical Industry Co., Ltd., Chuo-ku, Tokyo) and 10 mL of NGO suspension (0.5 mg/mL) were placed in sealed glass vial, sonicated for 2 h using a bath sonicator, and stirred for 4 h at room temperature. Then, co-products and excess reactants were removed from the suspension by washing 5 times with bicarbonate buffer (pH 8.5; 0.01 M). Alendronate trihydrate (16.26 mg; Sigma Aldrich, St. Louis, MO) was added to 10 mL of the suspension obtained, sonicated for 30 min and stirred overnight. Finally, NGO-ALs were purified by centrifuging at 27,230 g and reconstituting with distilled water ten times. Characterization of NGO-ALs Physiochemical characterizations The morphologies and sizes of NGO-ALs were determined by transmission electron microscopy (TEM). Briefly, a drop of an NGOAL suspension (0.01 mg/mL) was placed on a carbon-coated copper grid, dried at room temperature, and observed under a transmission electron microscope (H7600; Hitachi, Tokyo) at an accelerating voltage of 100 kV. FT-IR (Fourier transform-infrared) spectra of formulations were obtained using a Thermo Scientific Nicolet Nexus 670 FT-IT Spectrometer and Smart iTR (Thermo Fisher Scientific Inc., Waltham, MA) equipped with a diamond window.
Fig. 1. Bone-targeted delivery of DOX for treating bone metastasis from breast cancer. (A) Schematic illustration showing the construction of DOX@PEG-NGO-AL complexes. (B) Targeted delivery of DOX to bone tumor microenvironment by strong chelation of AL with hydroxyapatite and pH-responsive release of DOX from DOX@PEG-NGO-ALs.
Phosphate assay Amounts of AL that conjugated with NGOs were determined using a phosphate assay [26]. Briefly, 50 mL of an NGO-AL suspension (1 mg/mL), 3 mg of magnesium nitrate and 50 mL ethanol was added in a glass tube and heated over a strong flame until brown fumes disappeared. The ash was then treated with 0.3 mL hydrochloride acid (0.5 N; Sigma Aldrich, St. Louis, MO) at 100 C for 15 min, and then with 0.6 mL ammonium molybdate tetrahydrate (0.42% w/v in 1 N H2SO4; Sigma Aldrich, St. Louis, MO) and 0.1 mL ascorbic acid (10%, w/v; Sigma Aldrich, St. Louis, MO) at 37 C for 15 min. Solution absorbances were measured at 820 nm, and AL concentrations were read off standard calibration curves. NGO suspension was used as the negative control. Loading of DOX onto NGO-ALs
DOX loadings greater than those possible using other carriers [22]. Furthermore, DOX release from NGOs has been showed to be pHdependent [22–24], which suggests drug release would be accelerated in the acidic microenvironments of bone tumors and those created by resorption, and that DOX@PEG-NGO-AL nanocomposites offer promise for the treatment of bone metastasis. This study was undertaken to determine whether PEG-NGO-ALs enhance DOX accumulation in metastatic bone tumors in advanced breast cancer. Materials and methods
To incorporate DOX with NGO-ALs, 10 mL of doxorubicin hydrochloride solution (0.5 mg/mL) was added to 10 mL of NGOAL suspension (1 mg/mL). The mixture was then sonicated for 1 h and stirred overnight at room temperature. Free DOX was removed by repeated centrifugation at 5640 g using an ultracentrifugal filter tube (MWCO: 300 kDa; Vivaproducts Incs, Littleton, MA). Drug loading efficiencies (LEs) and drug loading capacities (LCs) were indirectly determined by measuring the concentration of free DOX in the filtrates. The concentration of DOX was quantified by UV–Vis absorbances at 481 nm using a microplate reader (Spark 10M; Tecan Australia, Port Melbourne, VIC, Australia).
Synthesis of NGOs Grafting of DSPE-PEG onto NGO-ALs Graphene oxide sheets (GOs) were prepared by modified Hummer’s methods using flake expandable graphite (Alfa Aesar, Thermo Fisher Scientific, Waltham, MA), as previously described [25]. Then, 10 mL of GO suspension (1 mg/mL) was then sonicated at 700 W for 2 h using a probe sonicator (VCX 750; Sonic1, Sonics & Materials, Inc, Newtown, CT) to produce exfoliated NGOs. Large GO sheets in suspension were further removed by centrifugation at 9425 g for 10 min.
To increase the stability of NGO derivatives under physiological conditions, DSPE-PEG (MW: 5000 Da; Nanocs, Boston, MA) was grafted onto NGOs, DOX@NGOs, NGO-ALs, and DOX@NGO-ALs by simply mixing DSPE-PEG with NGO-ALs in distilled water for 6 h. The stabilities of NGO-AL formulations with and without DSPEPEG were evaluated in phosphate buffer saline (PBS; 1X), fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT), or DMEM
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containing 10% FBS. In brief, formulations were added to media to a final concentration of 0.1 mg/mL and incubated at 37 C. After 24 h of incubation, the appearance of aggregation in each sample was assessed. The detachment of DSPE-PEG from nanosheets was investigated by using Cy5.5-labeled DSPE-PEG (Cy5.5-DSPE-PEG; MW: 5000 Da; Biochempeg, Watertown, MA). Briefly, DSPE-PEGgrafted formulations suspended in PBS, FBS, or DMEM containing 10% FBS were incubated at 37 C for 48 h. At predetermined times, samples were taken and centrifuged at 27,230 g for 1 h. The fluorescence intensities of free Cy5.5-DSPE-PEG in media were then measured using a microplate reader (Spark 10M; Tecan Australia; Port Melbourne, VIC, Australia). In vitro release study The release of DOX from DOX@PEG-NGO-ALs was assessed in phosphate buffer solution (PBS; pH 7.4) and acetate buffer solution (ABS; pH 4.5) by dialysis. A DOX weight equivalent of 0.1 mg of DOX@PEG-NGO-ALs was placed in a sealed dialysis bag (MWCO: 3500 Da; Spectra/Por, Spectrum Laboratories Inc., Rancho Dominguez, CA). The bag was then placed in a 50-mL conical tube containing 20 mL of either PBS or ABS. At regular time intervals, samples were withdrawn for analysis. Volume replenishment was performed using equal volumes of fresh medium. The concentrations of DOX in the release media were determined by UV–Vis spectrophotometry as described above. In vitro hydroxyapatite binding assay Hydroxyapatite (HA) microparticles (Sigma Aldrich, St. Louis, MO) were used to evaluate the affinity of NGO-ALs for bone. For this purpose, HA microparticles (20 mg) were mixed with 1 mL of either NGOs or NGO-ALs (0.1 mg/mL) in microtubes, and shaken for 4 h. At predetermined intervals (0.5 h, 1 h, 2 h, and 4 h), HA microparticles were allowed to settle, and supernatants were withdrawn for analysis. The UV absorbances of suspensions at 234 nm before (Abbefore) and after (Abafter) mixing with HA recorded using a UV–Vis spectrophotometer (Perkin Elmer U2800; Hitachi, Tokyo, Japan) were used to calculate the concentration of nanosheets. Weights of nanosheets bound to HA microparticles (Wb) were calculated using the following formula. Wb ðmgÞ ¼
Cbefore Cafter 100 Cbefore
Cbefore refers to the concentration of samples before mixing with HA powder, Cafter refers to the concentration of samples after mixing with HA powder. Similarly, decreases in fluorescence intensity at excitation and emission wavelengths of 673 nm and 797 nm, respectively, were used to examine the affinity of Cy5.5-labeled PEG-NGO-ALs for HA. Furthermore, HA microparticles in the pellets were washed 5 times with distilled water, freeze-dried, and observed under a scanning electron microscope (S-4100; Hitachi, Tokyo, Japan). Cell culture and growth condition MDA-MB-231 cells (a human breast carcinoma cell-line) were obtained from the Korean Cell Line Bank (Jongro-gu, Seoul, South Korea). Cells were cultured in DMEM containing 0.45% (w/v) glucose, 10% (v/v) FBS (Hyclone Laboratories, Logan, UT) and 1% (v/ v) penicillin-streptomycin (Hyclone Laboratories, Logan, UT). Cell viability assay The cytotoxic effects of NGOs, free DOX, PEG-NGOs, PEG-NGOALs, DOX@PEG-NGOs, and, DOX@PEG-NGO-ALs on MDA-MB-231 cells were evaluated using a cell counting kit-8 (CCK-8; Dojindo
Molecular Technology Inc., Rockville, MD) assay. Briefly, cells were cultured in a 96-well plate (Corning, Steuben County, NY) at a density of 5 103 cells/well. Samples were added to wells; the same volumes of PBS were added as negative controls. Following treatment for 24 h, cells were washed twice with PBS. The medium in each well was then replaced by an equivalent volume of fresh medium. WST-8 [10 mL; 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoium, monosodium salt] solution was then added to each well, and the plate was placed in a cell incubator for 3 h. After that, 70 mL of medium in each well was transferred into another 96-well plate for absorbance measurement at 450 nm using a plate reader (Spark 10M; Tecan Australia, Port Melbourne, VIC, Australia). Relative cell viabilities were calculated using the following formula. Cell viability ð% of controlÞ ¼
Absample Abblank 100% Abcontrol Abblank
Absample refers to the groups of cells treated with samples. Abcontrol refers to the groups treated with PBS. Abblank refers to medium only. Effect of DOX@PEG-NGO-ALs on an in vitro bone metastasis model An in vitro model, in which MDA-MB-231 cells were cultured in a three-dimensional collagen gel containing HA particles, was used to assess the effect of DOX@PEG-NGO-ALs on bone metastasis from breast cancer. Briefly, a bovine collagen solution (2.305 mL; 3 mg/mL; PureCol1, Advanced BioMatrix, CA) was mixed with a 20X PBS solution (0.188 mL), a sodium hydroxide solution (0.32 mL; 0.1 M), and distilled water (0.188 mL) at 4 C. Next, 10 mg of HA particles and 5 103 MDA-MB-231 cells were gently suspended in 100 mL of obtained collagen solution. The suspension was then transferred to a 96-well plate, which was pre-incubated at 40 C for 2 h. The plate was placed in a cell incubator for 3 h for complete gelation. The gel was washed twice with PBS and flipped using a sterile pipette tip to expose the HA surface. To evaluate the anti-tumor effect of DOX@PEG-NGOs and DOX@PEG-NGO-ALs, 100 mL of DOX@PEGNGOs and DOX@PEG-NGO-ALs suspension (equivalent to 25 mg DOX/mL) was added to the gels and incubated for 30 min under slight agitation. Same volumes of PBS were added as negative controls. The supernatant in each well was then removed and the gels were washed 3 times with PBS. The gels were incubated in 100 mL of culture media for 24 h. Finally, the cell viability was assessed using CCK-8 assay as described in Section “Cell viability assay”. In vivo biodistribution study The in vivo biodistribution of PEG-NGOs and PEG-NGO-ALs were investigated in immunocompromised BALC/c nude mice (Male; Orient-Bio Inc., Joongwon, Seongnam, South Korea). PEGNGOs and PEG-NGO-ALs were fluorescently labeled using DSPEPEG-Cy5.5. Sample stabilities in physiological salt buffer and affinities for HA were confirmed before administration. For wholebody imaging, 100 mL of a suspension of either PEG-NGOs or PEGNGO-ALs (2 mg/mL) was injected intravenously, and the distribution of fluorescence was recorded at day 1 and day 3 after injection using an in vivo imaging system (IVIS). The accumulation of nanosheets in the skeletal system was further assessed using an IVIS SpectrumCT in vivo imaging system (Caliper Lifescience, Hopkinton, MA). The distribution of fluorescence was analyzed based on a reconstructed three-dimensional diffuse tomography using Live Image1 3.1 software. To evaluate the selective delivery of nanosheets to bone tumors, an animal model of breast cancer bone metastasis was developed by injecting human breast adenocarcinoma MDA-MB-231 cells into the tibias of BABL/c nude mice, as previously reported [27].
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Briefly, mice were anesthetized with ketamine (80 mg/kg; Huons, Gyeonggi, South Korea) and xylazine (16 mg/kg; Bayer Korea Ltd., Seoul, South Korea), and a 31-gauge needle was then pushed through the patellar ligament into the tibia. A cell suspension (10 mL; 107 cells/mL) was then slowly injected into the marrow cavity. After 5 weeks, mice with a bone tumor were selected for in vivo distribution study and injected with Cy5.5-labeled nanosheets (100 mL; 2 mg/mL) via a tail vein. Whole body and tibia fluorescence intensities were recorded after 24 h using a fluorescently-labeled organism bioimaging instrument (FOBI; Neoscience, Suwon, Gyeonggi, South Korea). Statistical analysis Data were expressed as the mean standard deviation. The unpaired t-test or one-way ANOVA test was performed for the statistical analyses. P values < 0.05 were regarded as statistically significant. Results and discussion Characterization of NGOs GO flakes were prepared by chemically-induced oxidation of graphite using modified Hummer’s method [28]. FT-IR analysis confirmed the formation of new functional groups in GOs (Fig. S1A). A broad and intense peak at 3400 cm1 was ascribed to hydroxyl groups, a peak at approximately 1730 cm1 to the stretch of C¼O carboxylic moieties, and a peak at 1620 cm1 to aromatic C–C bonds. Peaks at 1365, 1215 and 1054 cm1 were ascribed to C–O–H deformation, C–H stretch (epoxy groups), and C–O stretch vibrations (alkoxy groups), respectively. NGOs were prepared by ultrasonication as previously described [29]; sonication time was optimized beforehand. After 2 h of sonication, mean dynamic particle sizes of GO sheets reduced
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significantly from approximately 750 nm to <200 nm (Fig. S1B). TEM revealed thin nanosheets with maximum dimensions of 60– 150 nm (Fig. 2A). FT-IR analysis confirmed functional groups of NGOs were not significantly altered by ultrasonication (Fig. S1B). UV–Vis spectrophotometry exhibited the characteristic peaks of NGOs, that are, a peak at 234 nm due to p–p* transition of aromatic C–C bonds and a shoulder at 300 nm due to n–p* transitions of C¼O bonds. Characterization of NGO-ALs NGOs were functionalized with AL due to covalent bond formation between the amine group of AL and the carboxylic acid groups of NGOs using EDC/NHS as cross-linkers. The FT–IR analysis revealed that the intensity of the peak at 1730 cm1 (corresponding to the carboxyl groups) of NGOs was significantly diminished, and that a new prominent peak related to stretching mode of amide groups appeared at 1631 cm1 (Fig. 2C), suggesting the formation of amide linkages between the carboxylic acid groups of NGOs and the amine group of AL. Additionally, two small peaks at 1020 cm1 and 926 cm1 appeared, which correspond to hydroxyl (–OH) group bending and P¼O stretching vibrations of AL. The amount of AL in NGO-ALs was 0.55 0.02 mg per 1 mg as determined in triplicated using phosphate assay. Loading and pH-triggered release of DOX DOX was non-covalently loaded onto NGO-ALs by simply mixing DOX and NGO-AL suspension with slight sonication. The formation of DOX@NGO-ALs was confirmed by UV–Vis spectrophotometry. As indicated in Fig. 2B, after removing free DOX, the characteristic absorption peaks of DOX at 233 nm and 480 nm were observed in the UV–Vis spectra of NGO-ALs, suggesting the stable incorporation of DOX. Notably, the DOX loading efficiency (LE) and loading capacity (LC) of DOX@NGO-ALs were 98.51 0.45% and 99.09 0.26%, respectively.
Fig. 2. Physiochemical characterization of DOX@PEG-NGO-ALs. (A) Transmission electron microcopy image of NGO-ALs. Scale bar: 100 nm. (B) UV–Vis spectra and optical image of DOX (red), NGO-ALs (blue) and DOX@NGO-ALs (green). (C) Fourier transform-infra red spectra of AL, NGO-ALs, DSPE-PEG, PEG-NGO-ALs, DOX, DOX@PEG-NGO-ALs. (D) Release profiles of DOX from DOX@PEG-NGO-ALs in acetate buffer (ABS; pH 4.5) and phosphate buffer (PBS; pH 7.4). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Szabó et al. reported that NGOs have extremely high surface areas (2600–2800 m2/g) [30], and thus, provide large surfaces for drug adsorption on edges and side of single atom thick two-dimensional NGO structures. This unique characteristic allows NGOs to possess remarkably higher LCs than other nano-carriers [31]. The high LE observed for DOX@NGO-ALs could be explained by strong p–p stacking interactions between the sp2-hybridized p bonds of NGO-ALs and the quinine portion of DOX and by hydrogen bonding between the carboxylic acid (–COOH), hydroxyl (–OH) groups of NGOs and the amine (–NH2), hydroxyl (–OH) groups of DOX. Interestingly, the release of DOX from DOX@NGO-ALs occurred in a pH-dependent manner. As indicated in Fig. 2D, only 16.87 2.84% of DOX was released after 60 h under neutral conditions (pH 7.4), whereas more than 60% of DOX was released at pH 4.5. This accelerated release under acidic conditions could be due to the partial protonation of the hydroxyl and amine groups of DOX, leading to higher drug solubility and weakening of hydrogen bonds between DOX and NGOs [30]. This pH stimuli-responsive behavior plays an important role in targeting effect of DOX@NGO-ALs since the acid–base balance in skeletal tissue is lost when bone metastasis occurs [32,33]. The predominant activity of osteoclasts in the tumor microenvironment increases the
concentration of H+ and acidic hydrolases in the bone resorption compartment and results in the digestion of the mineral and organic phases of bone matrix. Subsequently, pH values in osteolytic lesions formed by bone tumors decrease to approximately 4.5 [34]. Subsequenly, it is possible that slow release under neutral conditions and rapid release under acidic conditions may reduce the toxic effect of DOX on normal tissue and enhance anti-tumor efficiency. In vitro bone targeting The HA binding assay was conducted to evaluate the affinity of NGO-ALs for bone. HA microparticles were used as an in vitro model of bone because, as mentioned above, HA is the major mineral component of bone. As shown in Fig. 3B, NGO-ALs exhibited significantly higher affinity for HA than NGOs. After 1 h of mixing, more than 50% of NGO-ALs bound to HA microparticles, and this percentage gradually increased to approximately 70% at 4 h. In contrast, only 10.67% of NGOs bound to HA even after 4 h of mixing. In addition, the color of HA powder changed to grey and red after incubation with NGO-ALs and DOX@NGO-ALs, respectively, whereas no such change was observed for NGOs (Fig. 3C).
Fig. 3. In vitro hydroxyapatite binding assay. (A) Methodology to evaluate the affinity of NGOs and NGO-ALs to hydroxyapatite. (B) Affinity of NGOs and NGO-ALs to hydroxyapatite. (C) Digital image of hydroxyapatite powder after 4 h of mixing with NGOs, NGO-ALs and DOX@NGO-ALs. (D) SEM image of HA microparticles before and after 4 h of mixing with NGOs and NGO-ALs. Scale bar: 1 mm.
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Further assessment of the surface morphology of HA by SEM revealed dense accumulations of NGO-ALs on the surface of microparticles, while NGOs were barely observed on the surfaces of microparticles after mixing (Fig. 3D). These results indicate that NGO-ALs have a remarkably strong affinity for HA, suggesting that AL-functionalized NGOs offer a means of delivering drugs to bone. Incorporation of DSPE-PEG onto DOX@NGO-ALs NGOs aggregate in the presence of salts in physiological buffers due to the charge screening effect. To address this problem, researchers have surface-modified NGOs with hydrophilic polymers to improve their stabilities [35–38]. Similarly, in this study, PEG was used as the stabilizer due to its solubility and biocompatibility. DSPE-PEG was grafted hydrophobically onto NGO-ALs using the interaction between the lipid chain of DSPE and sp2-hybridized p-conjugated structure of NGOs. DSPE-PEG was added to nanosheet suspension of NGO-ALs at a weight ratio of 1:1 without any significant change in affinity for HA (Fig. S3A–B). Both
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PEG-NGO-AL and DOX@PEG-NGO-AL suspensions proved stable in PBS and DMEM containing 10% FBS for 24 h, whereas in the absence of DSPE-PEG, the agglomeration was clearly observed (Fig. S3C). In addition, we examined DSPE-PEG loss from these NGOs in three different media: PBS (pH 7.4), FBS, and DMEM containing 10% FBS. Less than 10% of Cy5.5-DSPE-PEG was found to be released after 48 h of incubation in all three media (Fig. S3D). These results suggest that DSPE-PEG might be an effective stabilizer for preventing DOX@NGO-AL aggregation under physiological conditions. Furthermore, the incorporation of Cy5.5-DSPE-PEG onto NGO-ALs provides a means for fluorescent-labeling nanosheets. In vitro cytotoxicity Cytotoxicities of DOX, PEG-NGO-ALs and DOX@PEG-NGO-ALs on MDA-MB-231 cell line were investigated using a CCK-8 assay. PEG-NGOs and PEG-NGO-ALs had no obvious toxic effect even at 100 mg/mL (Fig. 4A). In addition, DOX and DOX@PEG-NGO-ALs exhibited similar time-dependent toxic effects on MDA-MB-231
Fig. 4. In vitro anti-tumor effect of DOX@PEG-NGO-ALs. (A) Cytotoxicity of PEG-NGOs and PEG-NGO-ALs at concentration of 1.25 mg/mL, 6.25 mg/mL, 12.5 mg/mL, 25 mg/mL, 50 mg/mL, 100 mg/mL. (B) Concentration-dependent cytotoxicity of free DOX and DOX@PEG-NGO-ALs. (C) Anti-tumor effect of DOX@PEG-NGOs and DOX@PEG-NGO-ALs assessed using a co-culture model of MDA-MB-231 cells and hydroxyapatite particles in three-dimensional collagen gel.
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cell line (Fig. 4B), suggesting the incorporation of DOX with PEGNGO-ALs did not reduce its therapeutic potency. To further assess the potential of DOX@PEG-NGO-ALs for treating bone metastasis from breast cancer, we developed an in vitro bone tumor model, in which MDA-MB-231 cells were co-cultured with HA particles in a three-dimensional collagen gel (Fig. 4C). Since the physiological structure of bone mainly consists of HA and bone matrix proteins, collagen gel containing HA particles can relatively imitate the natural environment of bone for the growth of breast cancer cells. Interestingly, DOX@PEG-NGO-ALs exhibited significantly higher toxicity as compared to DOX@PEG-NGOs on MDA-MB-231 cells in our in vitro bone cancer model. This suggests the superiority of DOX@PEG-NGO-ALs for treating bone tumor as compared to nonfunctionalized DOX@PEG-NGOs. In vivo bone-homing effects To investigate the role of the AL component on the bonehoming ability of PEG-NGO-ALs, Cy5.5-labeled PEG-NGOs and Cy5.5-labeled PEG-NGO-ALs were injected into BALB/c nude mice via a tail vein. IVIS images revealed that PEG-NGO-ALs were
Fig. 5. In vivo biodistribution of PEG-NGOs and PEG-NGO-ALs on day 3 after intravenous injection.
retained in mice body longer than PEG-NGOs (Figs. 5A and S4), which concurs with previous studies [17,39]. In addition, PEGNGOs mostly accumulated in liver and spleen, whereas PEG-NGOALs were observed liver, spleen, femurs, and tibias. Similarly, Dhifaf et al. described that 24 h after injection, PEG-NGOs accumulated in organs of the reticuloendothelial system (RES), including liver and spleen, and eventually be eliminated by urinary excretion [40]. In another study, the blood circulation half-life of NGOs and PEG-NGOs were reported to be 5.35 h and 6.29 h, respectively [41]. After entering the blood stream, NGOs may tend to roll and fold to form smaller particles, and thus, are able to cross the glomerular filtration barrier [40,42,43]. On the other hand, larger NGOs may be uptaken by the RES and localize in liver and spleen [40,44]. This could be why PEG-NGOs accumulated in RES organs and fluorescence intensity rapidly diminished on days 1 and 3 post-injection in the PEG-NGOs treated mice. We speculate that the greater retention time of PEGNGO-ALs as compared with non-functionalized NGOs could be due to interaction with the skeletal system, and the confinement of nanosheets to bone might delay entrapment by the RES and accessibility to the glomerular filtration barrier, and thus, increase in vivo retention. The accumulation of nanosheets in the skeletal system on day 3 post-injection was examined by IVIS/ micro CT imaging (Fig. 5B). NGO-ALs were clearly observed in femurs, tibias, and ribs, whereas NGO fluorescence was barely found in the skeletal system. These results suggest that functionalization of NGOs with AL increases NGO accumulation in the skeletal system and enhances in vivo retention times after intravenous administration.
Fig. 6. The preferential localization of PEG-NGO-ALs at the site of bone tumor. (A) Optical image and fluorescence image of healthy tibiae and tibiae bearing tumor after 24 h post-injection of Cy5.5-labeled PEG-NGO-AL. (B) Quantitative measurement of fluorescence intensity in healthy tibiae and tibiae bearing tumor after 24 h post-injection of Cy5.5-labeled PEG-NGO-AL (n = 4). **p < 0.05.
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The different biodistributions of PEG-NGO-ALs in healthy bones and bone bearing tumors were also evaluated using a murine bone cancer model, in which the intra-tibial inoculation of MDA-MB231 cells was used to mimick the final stage of bone metastasis in breast cancer. Though the early metastatic steps are bypassed, this model provides similar tumor-takes and osteolytic lesion development rates among recipients [27]. In addition, this model enables the role played by the bone tumor microenvironment to be evaluated without concerns about interference by tumors in other sites. We assessed the fluorescence intensities of heathy tibias isolated from controls and tumor-bearing tibias 24 h after injection. As shown in Fig. 6, fluorescence intensities in tibiabearing tumors were significantly higher as compared to fluorescence intensities in the healthy tibia (p < 0.05; n = 4), indicating the preferential accumulation of nanosheets in bone cancer microenvironments. This phenomenon might be due to greater HA exposure in such regions. As previously reported, bone surfaces are generally covered by cells and organic matrix, which might reduce AL binding, whereas bone remodeling during osteolytic lesion development leads to the digestion of bone organic matrix and cell detachment [45,46], which would provide a more accessible surface for AL chelation. This selective accumulation suggests that PEG-NGO-ALs are viewed as a potential carrier for the targeted delivery of drugs to sites of bone metastasis. Conclusion In summary, this study describes a promising strategy for treating bone metastasis in advanced breast cancer using DOX@PEG-NGO-ALs. The DOX@PEG-NGO-AL formulation exhibited rapid DOX release under acidic condition which mimics the microenvironment of bone tumors. Most importantly, AL functionalization significantly increased the retention and accumulation of nanosheets in the murine skeletal system, especially in osteolytic lesions. These findings suggest PEG-NGO-ALs provide a potential strategy for enhancing antitumor effects and for diminishing the off-target toxicities of anticancer drugs used for the treatment of bone cancer. The unique ability of NGOs to carry and preserve the stabilities of different therapeutic agents, which include growth factors (BMP-2, VEGF), siRNAs, and plasmid DNAs, encourages us to believe that application of the PEG-NGO-ALs platform could be extended to the treatments of other bonerelated diseases, such as, osteoporosis and Paget’s disease. Declaration of interest The authors have no conflict of interest to declare. Acknowledgments This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Korean Ministry of Science, ICT, and Future Planning (grant no. 2015R1A5A2009124) and funded by the Ministry of Education (grant no. 2017R1D1A1B03027831); and by the Korea Health Technology R & D Project through the Korea Health Industry Development Institute (KHIDI) and the Korean Ministry of Health and Welfare (grant no. HI16C1767 and HI18C045 3); and by the Creative Economy Leading Technology Development Program through the Gyeongsangbuk-Do and Gyeongbuk Science and Technology Promotion Center of Korea (grant no. SF316001A).
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