Bone-targeted delivery of nanodiamond-based drug carriers conjugated with alendronate for potential osteoporosis treatment

Journal of Controlled Release 232 (2016) 152–160

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Bone-targeted delivery of nanodiamond-based drug carriers conjugated with alendronate for potential osteoporosis treatment Tae-Kyung Ryu a, Rae-Hyoung Kang a, Ki-Young Jeong a, Dae-Ryong Jun a, Jung-Min Koh b, Doyun Kim c, Soo Kyung Bae c, Sung-Wook Choi a,⁎ a b c

Department of Biotechnology, The Catholic University of Korea, 43 Jibong-ro Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea Division of Endocrinology and Metabolism, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 138-736, Republic of Korea College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea

a r t i c l e

i n f o

Article history: Received 5 November 2015 Received in revised form 12 April 2016 Accepted 15 April 2016 Available online 17 April 2016 Keywords: Nanodiamond Alendronate Bone targeting Osteoporosis

a b s t r a c t This paper describes the design of alendronate-conjugated nanodiamonds (Alen-NDs) and evaluation of their feasibility for bone-targeted delivery. Alen-NDs exhibited a high affinity to hydroxyapatite (HAp, the mineral component of bone) due to the presence of Alen. Unlike NDs (without Alen), Alen-NDs were preferentially taken up by MC3T3-E1 osteoblast-like cells, compared to NIH3T3 and HepG2 cells, suggesting their cellular specificity. In addition, NDs itself increased ALP activity of MC3T3-E1 cells, compared to control group (osteogenic medium) and Alen-NDs exhibited more enhanced ALP activity. In addition, an in vivo study revealed that AlenNDs effectively accumulated in bone tissues after intravenous tail vein injection. These results confirm the superior properties of Alen-NDs with advantages of high HAp affinity, specific uptake for MC3T3-E1 cells, positive synergistic effect for ALP activity, and in vivo bone targeting ability. The Alen-NDs can potentially be employed for osteoporosis treatment by delivering both NDs and Alen to bone tissue. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Multi-functional nanoparticles have been of great importance in various biomedical applications, such as delivery systems [1,2], biosensors [3], bioimaging [4,5], and tissue engineering [6]. Among the many materials for nanoparticles, carbon-based materials have extended applications in biomedical engineering, including nanodiamonds (NDs), graphene, carbon nanotubes, fullerenes, carbon nanofibers, and nanohorns [7]. Recently, NDs, which are carbon-based allotrope nanoparticles of truncated octahedral composition, have attracted much attention as a promising nanomaterial due to their spherical morphology, high surface functionality, high biocompability, and strong hardness [8]. Chow et al. demonstrated inhibited tumor growth using doxorubicin-conjugated NDs by overcoming the drug efflux issue [9]. Guan et al. fabricated ND-based vehicles containing cisplatin and demonstrated pH-responsive release for cancer treatment [10]. So far, most studies related to drug delivery using NDs have been limited to cancer treatment. Targeted drug delivery is considered a promising system capable of minimizing the side effects of therapeutic agents, particularly those of hormones. For bone targeted drug delivery, there are well-known functional ligands including bisphosphonate (BP), tetracycline [11],

⁎ Corresponding author. E-mail address: [email protected] (S.-W. Choi).

http://dx.doi.org/10.1016/j.jconrel.2016.04.025 0168-3659/© 2016 Elsevier B.V. All rights reserved.

Alizarin Red S, and small peptide aspartic acid [12]. Among them, BPs (ex, alendronate (Alen), risedronate, etidronate) are often used for bone targeted drug delivery due to their high affinity to bone and therapeutic effects on bone diseases. Yokogawa et al. demonstrated specific delivery of estrogen to bone using a peptide with glutamic acid and aspartic acid [13]. Wang et al. found a slightly higher in vivo binding efficiency of Alen to bone, compared to that of an aspartic acid peptide [14]. BPs are often used as ligands due to their therapeutic effects in various bone diseases (osteoporosis, Paget's disease, and metastatic bone cancer), as well as their high affinity to bone tissue [15,16]. Choi et al. prepared poly(D,L-lactide-co-glycolide) nanoparticles modified with Alen and polyethylene glycol and demonstrated strong binding to hydroxyapatite (HAp) and in vitro release of estrogen [17]. Recently, Zhang et al. fabricated ND-composited biodegradable scaffolds and demonstrated their enhanced proliferation and differentiation of osteoblast, suggesting the positive effect of NDs on osteoblasts [6]. Inspired this report, we designed therapeutic carriers delivering both NDs and drug specifically into bone tissue for potential applications in bone disease treatment. In this work, Alen was chosen as a targeting ligand due to the advantage of easy conjugation to carboxyl groups at the surface of NDs. The rationale for selecting NDs as a carrier material was based on their high alkaline phosphatase (ALP) activity as well as the high surface functionality, biocompatibility, spherical morphology, and hardness. The advantages of NDs in targeted delivery include their spherical morphology and high surface functionality

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which facilitates rapid cellular uptake and employment of various and many types of functional ligands [8,18,19]. Herein, we designed Alen-conjugated NDs and evaluated their potential for bone targeted delivery by specifically delivering both biocompatible functional nanomaterials (NDs) and therapeutic agent (Alen) into bone tissue. We conjugated Alen and/or dye onto the ND surface and systematically evaluated their affinity to hydroxyapatite (HAp), cellular uptake behavior, and in vivo targeting ability, compared to dye-conjugated Alen. To our knowledge, this is the first in vitro and in vivo demonstration of bone-targeted delivery based on NDs. 2. Materials and methods 2.1. Materials The NDs were supplied from Real-Derzinski, Co. (Russian Federation). Alen was kindly provided by Samjin Pharm. Co. Ltd (Seoul, Korea). Dimethyl sulfoxide (DMSO), N-hydroxysuccinimide (NHS), ethyl(dimethylaminopropyl)carbodiimide (EDC), poly(D,Llactide-co-glycolide) (PLGA, L/G = 75/25, MW = 66.000–107,000), pyrene, fluorescein 5(6)-isothiocyanate (FITC), and rhodamine B isothiocyanate (RITC) were purchased from Sigma-Aldrich (St Louis, MO, USA). The dialysis membranes with MWCO values of 3.5 and 12–14 kDa were purchased from Spectrum Laboratories Inc. (Rancho Dominguez, CA, USA). Alexa Fluor 546 phalloidin, phosphate buffered saline pH 7.4 (PBS), and culture media were purchased from Invitrogen (Grand Island, NY, USA). The culture medium consisted of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (containing penicillin and streptomycin). The Cell Counting Kit-8 (CCK-8, Dojindo Co. Ltd., Tokyo, Japan) was used to quantitatively measure cell number. The Bio-Rad Protein Assay kit was purchased from Bio-Rad Laboratories (Hercules, CA, USA). 2.2. Conjugation of Alen to NDs As reported previously [20], ND dispersion in acetone (0.1 wt.%) were obtained by treating with oleic acid. ND nanoparticles (NDs) with approximately 50 nm in size were fabricated by using a simple fluidic device [21]. For conjugation, amine-terminated FITC was synthesized by reacting FITC (3.2 mg) and ethylenediamine (5.5 μL) at a mole ratio of 1:10 in 1 mL of DMSO for 12 h at room temperature, followed by dialysis and then freeze-drying [22]. FITC and Alen were conjugated to the NDs by carbodiimide chemistry with EDC and NHS [23]. EDC (383.4 mg) and NHS (230.0 mg) were added to the ND dispersion (0.1 wt.%, 20 mL) in water and stirred at room temperature for 12 h to activate the carboxyl groups. The NDs in water (20 mL) were conjugated with amine-terminated FITC (2.0 mg) to obtain FITC-conjugated ND nanoparticles (NDs) and then with Alen (10 mg) to obtain FITCand Alen-conjugated ND nanoparticles (Alen-NDs). For comparison, FITC-conjugated Alen was prepared as a macromolecular conjugate by reacting Alen (10 mg) and FITC (2.0 mg) under the same conditions as those for the amine-terminated FITC. After 3 days of dialysis, Alen, NDs, and Alen-NDs were analyzed with a Zeta potential/ size analyzer (Malvern Instruments Ltd., Worcestershire, UK), and a UV/vis spectrophotometer (Perkin Elmer, Norwalk, CT, USA). Surface morphology was characterized by a scanning electron microscopy (SEM; Hitachi, Tokyo, Japan). To evaluate the stability in serum, the NDs and Alen-NDs were dispersed in PBS (pH 7.4) containing 20% fetal bovine serum and their Zeta-potential and size were monitored for 14 days.

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filter (0.22 μm pore size), absorbance of the filtrates was determined by a UV/vis spectrophotometer at 488 nm (characteristic FITC peak), corresponding to the unbound amount of sample to HAp. The binding ratio to HAp was defined as the reduction in percent absorbance at 488 nm. 2.4. Conjugation stability of Alen-NDs To evaluate conjugation stability between Alen and NDs, the AlenNDs (3 mg/mL) were incubated in PBS (pH 7.4) at 37 °C or aqueous HCl solution (6 M, control) at 90 °C. At the predetermined time points, the samples (0.2 mL) were withdrawn and then centrifuged at 5000 rpm for 10 min in ultrafiltration tubes (Amicon Ultra, MWCO 30 kDa, Merck Millipore, Billerica, MA, USA). The concentration of free Alen degraded from Alen-NDs in the ultrafiltrate was measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with API 5500 Q-Trap mass spectrometer (AB SCIEX, Foster City, CA, USA) and a 1260 HPLC system (Agilent Technologies, Wilmington, DE, USA) [24]. All experiments were carried out in triplicates. Furthermore, the conjugation stability of Alen-NDs within cells was evaluated by analyzing the concentration of free Alen after cellular uptake of Alen-NDs. MC3T3-E1 cells (5 × 104 cells per well) were seeded on 24-well culture plates and incubated with Alen-NDs. At predetermined time points, the cells were washed three times with PBS to remove non-uptaken Alen-NDs and harvested from the culture plates using trypsin. The cell dispersions were centrifuged for 3 min at 1200 rpm, obtaining cell pellets. The cycle of freezing and thawing was repeated three times to destroy the cell membrane and then the concentration of free Alen was measured using the LC-MS/MS method after the centrifugation. 2.5. Cellular uptake Human hepatoma (HepG2), mouse embryonic fibroblast (NIH3T3), and mouse calvaria-derived pre-osteoblast (MC3T3-E1) cells were purchased from the Korea Cell Line Bank (Seoul, Korea). Each cell line was seeded on 24-well culture plates at a concentration of 1 × 104 cells per well and cultured in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin and streptomycin at 37 °C in 5% CO2 and 95% air. After 24 h, each well was washed three times with PBS and replaced with new culture medium. NDs and Alen-NDs (0.1 mL, 0.1 wt.% in PBS) were added to the wells containing each cell line. At predetermined time (3 and 12 h), each well was washed three times with PBS to remove the NDs and Alen-NDs that were not taken up by the cells. To quantitatively measure the amount of cellular uptake, cells in each well were solubilized with 200 μL HCl for 24 h and transferred to a 96-well plate to measure the amounts of NDs and Alen-NDs using a microplate reader (Molecular Devices, Co. Ltd., Sunnyvale, CA, USA) at 488 nm. Cell number per well was determined using the CCK-8 assay according to the manufacturer's instructions. The amount of cellular uptake (pg/cell) was calculated as the amounts of NDs and Alen-NDs divided by the number of cells. In addition, MC3T3-E1 cells treated with NDs and Alen-NDs were washed three times with PBS and fixed in 4% paraformaldehyde (500 μL) at room temperature for 3 h, followed by washing three times with PBS. F-actin (red) and cell nuclei (blue) were stained with Alexa Fluor 546 phalloidin and 4-6-diamidino-2-phenylindole (DAPI), respectively. Cell morphology was observed under a confocal laser scanning microscope (Nikon, Tokyo, Japan). 2.6. ALP activity assay

2.3. HAp affinity assay To evaluate the affinity to HAp, Alen, NDs, and Alen-NDs (5 mL, 0.1 wt.%) were separately added to a HAp dispersion (10 mg/mL) in PBS and shaken for 1 h in the dark. After filtration through a syringe

After 1, 5, and 7 days of culture, alkaline phosphatase (ALP) activity was measured as described previously with some modifications. In brief, MC3T3-E1 cells were seeded at a density of 1 × 104 cells in 24-well plates and then incubated with Alen, NDs, and Alen-NDs

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(n = 3 per group per time point). The concentration of Alen in Alen-NDs was adjusted to the same as the Alen group. The cells were washed three times with PBS and then lysed in 1 mL alkaline lysis buffer containing 0.1 M glycine, 1 mM MgCl2, and 0.05% Triton X-100. The cells in lysis buffer were centrifuged at 13,000 rpm at 4 °C for 10 min to remove cell debris, and the supernatants were stored at −20 °C for 30 min. p-Nitrophenyl phosphate (pNPP) solution (1 mg/mL) in 0.2 M Tris-buffer was prepared from SIGMAFAST pNPP tablets (Sigma) according to the manufacturer's instructions. Reactions were carried out in a 96-well plate by adding cell lysate (50 μL) and pNPP (50 μL) solution to each well, and the plate was kept at 37 °C for 30 min. ALP activity was determined by measuring conversion of p-NPP to p-nitrophenol. Then, the reaction was stopped by adding 3 N NaOH (50 μL), and absorbance was measured immediately using a microplate reader at a wavelength of 405 nm. ALP activity was normalized to total protein content. Total protein content was measured using the Bio-Rad Protein Assay kit. In the protein assay, the protein solution (200 μL), cell lysate (2 μL), and distilled water (800 μL) were added to each well. The absorbance of the protein solution at was measured at a wavelength of 595 nm using a microplate reader. To evaluate the effects of Alen concentration on cell toxicity and cellular differentiation, Alen concentrations were adjusted to 0.001– 100 mg/mL. After adding aqueous solutions (0.1 mL) with different Alen concentrations to each well, cell toxicity was measured and the ALP assay was conducted after 24 h incubation. Untreated cells served as a negative control. The cell toxicity was determined using the CCK8 according to the manufacturer's instructions and calculated as the number of dead cells divided by the number of total cells. 2.7. Performance comparison with PLGA-based nanoparticles To compare performances of ND-based delivery system, PLGA nanoparticles were prepared using a simple fluidic device and nanoprecipitation method [21]. PLGA solution in acetone (0.5 wt.%) was introduced into the fluidic device as the discontinuous phase, where water without any surfactant served as the continuous phase. The flow rates of the discontinuous and continuous phases were kept at 0.02 and 1 mL/min, respectively. The precipitated PLGA nanoparticles at the tip of the needle flew into the collection phase (same as the continuous phase) under gentle magnetic stirring. PLGA nanoparticles were obtained after acetone evaporation. The PLGA nanoparticles in water were conjugated with Alen using carbodiimide chemistry with EDC and NHS [23], obtaining Alen-conjugated PLGA (Alen-PLGA) nanoparticles. The average sizes of PLGA and Alen-PLGA nanoparticles were analyzed with a Zeta potential/size analyzer. For quantification of PLGA-based nanoparticles, pyrene (0.05 wt.%) was added to PLGA solution (0.5 wt.% in acetone) prior to preparation. The PLGA nanoparticles containing pyrene was analyzed using UV/vis spectrophotometer. For cellular uptake behavior, MC3T3-E1 cells were seeded at a density of 1 × 104 cells in 24-well plates and treated with NDs, PLGA, Alen-NDs, and Alen-PLGA. The concentrations of each type of the nanoparticles were adjusted to 0.1 wt.% (0.1 mL). At 3 and 12 h, the amount of cellular uptake was determined using a microplate reader at 264 nm for PLGA-based nanoparticles and at 488 nm for NDs-based nanoparticles (conjugated with FITC). Cellular uptake ratio (%) was calculated as the amount of cellular uptake divided by total amount of the nanoparticles. In addition, ALP activity was evaluated as described above after treatment of MC3T3-E1 cells with PLGA, Alen, and Alen-PLGA nanoparticles dispersed in osteogenic medium. The concentration of Alen in Alen-PLGA was adjusted to the same as the Alen group. 2.8. In vivo animal study RITC was used instead of FITC to avoid overlap with fluorescence of the phosphor [25]. Amine-terminated RITC was prepared using the

same procedure as that for amine-terminated FITC and then used for further conjugation with Alen and NDs. Female nude mice (4 weeks, BALB/c, nu/nu mice; Orient-Bio, Seoul, Korea) were randomly divided into four groups of RITC, RITC-conjugated Alen, RITC-conjugated NDs, and RITC-conjugated Alen-NDs. The RITC concentration for each sample was kept constant (1.8 × 10−5 mol/kg). The four samples were injected into the nude mice through the tail vein after anesthetization with Zoletil 50® (10 mg/kg, ip; Vibac Laboratories, Carros, France). Animals in each group were observed 12 h post injection using Image Station 4000 MM (Kodak, Danbury, CT, USA) equipped with a special C-mount lens and a long wave emission filter (535–600 nm; Omega Optical, Brattleboro, VT, USA). After euthanizing the animals with carbon dioxide gas, the livers, lungs, spleens, kidneys, hearts, and vertebra were extracted and photographed using a 12-bit CCD camera. Vertebra was chosen as a representative bone due to the advantage for harvesting and imaging. The explanted vertebra was fixed in formalin, dehydrated in acetone, and sectioned to 100 μm thickness with a microtome (Cryotome FSE; Thermo Scientific, Co. Ltd., Rockford, IL, USA). The slices were mounted on a plastic coverslide for observation under a fluorescence microscope (IX71; Olympus, Tokyo, Japan). To quantitatively determine the amount of sample bound to mouse bone tissue, all bone was crushed with a mortar in water and sonicated using a Vibra Cell™ instrument (Sonics and Materials Inc., Danbury, CT, USA) for 3 min at 110 W in an ice bath. The bone powder dispersions were centrifuged at 20,000 rpm for 10 min, and absorbance of the supernatant was measured at 510 nm (characteristic RITC peak) using a microplate reader. All experiments including live animals were performed in accordance with the guidelines of an approved protocol from the Institutional Animal Care and Use Committee of The Catholic University of Korea (Republic of Korea). 2.9. Statistics All data are expressed as mean with standard deviation (mean ± SD). Statistical comparisons were evaluated by analysis of variance (ANOVA), and statistical significance was accepted at p b 0.05. 3. Results and discussion 3.1. Preparation and characterization of Alen-conjugated NDs The pristine NDs prepared by detonation method easily aggregate to form large clusters due to their poor dispersibility [26–28]. Therefore, NDs modified with oleic acid were used in this work [20]. A simple fluidic device was employed to control the size of NDs [21], where ND dispersion in acetone was introduced as the discontinuous phase and water without any surfactant served as the continuous phase. NDs with approximately 50 nm in size were finally obtained after acetone evaporation. The resultant NDs exhibited a low cytotoxicity (Fig. S1). Neither the Alen nor NDs in water had a characteristic UV/vis spectral peak (Fig. S2). Therefore, FITC was conjugated to Alen and NDs for quantification and visualization. Fig. 1 shows the characteristics of the FITC-conjugated Alen, NDs, and Alen-NDs. The average size of NDs was adjusted to approximately 50 nm due to their high cellular uptake efficiency [21]. As shown in Fig. 1A, NDs and Alen-NDs had average sizes of 50.53 ± 12.15 and 64.74 ± 20.76 nm, as measured by dynamic laser light scattering. Both NDs and Alen-NDs had relatively narrow size distributions without tails N100 nm. In addition, the pore size (80–100 nm) of bone vasculature is another reason for controlling the size of NDs for bone-targeted delivery [29]. The slight difference in size could be attributed to aggregation during conjugation. Alen and NDs in PBS at pH 7.4 exhibited negative charges with zeta potentials of −16.0 and −40.3 mV, respectively (Fig. 1B). A previous study reported that NDs modified with oleic acid were well dispersed in oil due to hydrogen bonding between carboxyl

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Fig. 1. (A) Size distributions, (B) Zeta-potentials, and (C) UV/vis spectra of Alen, NDs, and Alen-NDs. (D) SEM image of Alen-NDs.

groups on the surface of the pristine NDs and oleic acid [30]. Alen-NDs also had a high negative charge (− 39.0 mV), suggesting good longterm colloidal stability. Both NDs and Alen-NDs exhibited characteristic FITC peaks in the UV/vis spectra (Fig. 1C), indicating FITC conjugation. Fourier transform infrared spectra also confirmed successful conjugation (Fig. S3). Fig. 1D shows a SEM image of Alen-NDs with a spherical morphology. Zeta-potential and size of both NDs and Alen-NDs were only slightly changed in PBS containing 20% fetal bovine serum for 14 days (Fig. S4), suggesting the ND-based nanoparticles could maintain their stability in the bloodstream. Alen reduces periprosthetic bone loss by suppressing osteoclastic activation during osteoporosis treatment [31]. In addition, Alen is known to induce osteoblast differentiation, leading to an increase in ALP activity [32]. However, a high concentration of Alen (N10− 5 M) has significant cytotoxicity [33]. Therefore, the effects of Alen concentration on ALP activity and cell viability were evaluated to maximize therapeutic function. ALP activity was maintained at b0.1 mg/mL Alen, whereas cell toxicity increased gradually (Fig. 2). Above 0.1 mg/mL, the increase in Alen concentration led to a significant decrease in ALP activity and increase in cell toxicity. This result suggests that 0.1 mg/mL is the optimal concentration. Therefore, the Alen

Fig. 2. Variations in alkaline phosphatase (ALP) activity and cell toxicity with respect to Alen concentration.

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concentration in the Alen conjugates was adjusted to 0.1 mg/mL for subsequent experiments. The concentration of NDs was adjusted to that of the NDs in Alen-NDs. 3.2. HAp affinity High affinity to bone tissue is the first requirement for bone-targeted delivery. Therefore, the affinity of Alen-NDs to HAp was quantitatively analyzed using HAp powder as model bone. Fig. 3 shows the binding ratio of Alen, NDs, and Alen-NDs to HAp, where FITC served as control. Most (77.50%) of the Alen bound to the HAp powder, whereas only small amounts of FITC (3.59%) and NDs (3.20%) were non-specifically adsorbed. Alen-NDs exhibited a lower binding ratio (36.73%) than that of Alen, which was attributed to the larger size of Alen-NDs than that of Alen molecules. Despite the lower binding ratio of Alen-NDs, this result confirms the favorable binding affinity of Alen-NDs to bone tissue. Previous researchers have demonstrated that binding of macromolecular conjugates (based on Alen or acidic peptide) onto HAp quickly occurs and that the binding amount does not change significantly over time in vitro or in vivo [14,34]. This quick and strong affinity of Alen conjugates makes our approach based on hard NDs plausible. Once Alen-NDs are bound to bone, they remain and are incorporated into bone tissue for a long period of time, possibly increasing mechanical strength of the bone similar to ND-composited scaffolds. 3.3. Conjugation stability The conjugation stability between Alen and NDs in Alen-NDs was evaluated by measuring the concentration of free Alen degraded from Alen-NDs after incubation in PBS (pH 7.4) at 37 °C or aqueous HCl solution (6 M, control) at 90 °C [35]. As shown in Fig. 4, the concentration of free Alen in aqueous HCl solution was increased over time due to hydrolysis of amide bond and reached 805.5 μg/mL at 24 h. However, only a small amount (1.03 μg/mL) of Alen was degraded from Alen-NDs in PBS at 24 h, suggesting the stability of the conjugation in PBS. After Alen-NDs treatment, the concentration of free Alen within MC3T3-E1 cells also maintained in the range of 0.25–0.27 μg/mL for 24 h (Fig. S5). These results confirmed that the conjugation in Alen-NDs was stable in both PBS and cells. Therefore, Alen-NDs might affect to the bone-related cells in the conjugated form, not degraded one. 3.4. Cellular uptake and ALP assay

Fig. 4. Variation of the concentration of free Alen degraded from Alen-NDs in PBS (pH 7.4) and aqueous HCl solution (6 M) with respect to time (n = 3). The error bars in PBS are hidden behind the data symbols.

conjugated Alen to evaluate the specific cellular association of Alen. The cellular uptake amount of Alen onto MC3T3-E1 cells was significantly greater than those onto HepG2 and NIH3T3 cells even at 3 h incubation (Fig. S6), suggesting the specific affinity of Alen onto MC3T3-E1 cells [15,36,37]. To evaluate specific cellular uptake, NDs and Alen-NDs were incubated with HepG2, NIH3T3, and MC3T3-E1 for 3 and 12 h. As shown in Fig. 5, little difference in the amount of cellular uptake was detected between NDs and Alen-NDs in the three types of cells at the initial stage (3 h). After 12 h of incubation, the amount of cellular uptake increased in the NDs and Alen-NDs in three types of cells. Note that a significantly larger amount of Alen-NDs was taken up by MC3T3-E1 cells among the cell types, whereas similar amounts of NDs were taken up by the three types of cells. This result revealed the specific uptake of Alen-NDs into MC3T3-E1 cells, which might be due to preferential affinity of BPs to protein tyrosine phosphatases of osteoblast [36]. Fig. 6 shows the confocal microscopy images of MC3T3-E1 cells incubated with NDs and Alen-NDs for 3 and 12 h. In the case of NDs, very low fluorescence was observed in MC3T3-E1 cells even at 12 h, suggesting no preferential affinity between NDs and MC3T3-E1 cells. Note that most Alen-NDs were observed inside the cells at 12 h. The

Prior to cellular uptake experiment of NDs and Alen-NDs, three types of cells (HepG2, NIH3T3, and MC3T3-E1) were treated with FITC-

Fig. 3. Plot of binding ratio of Alen, NDs, and Alen-NDs to HAp powder, where aqueous FITC solution served as the control. Data are mean ± standard deviation (n = 3). ⁎Significant difference between the two groups (p b 0.05).

Fig. 5. Plots of cellular uptake amount of NDs and Alen-NDs into HepG2, NIH3T3, and MC3T3-E1 cells after 3 and 12 h incubations. Data are mean ± standard deviation (n = 3). ⁎Significant difference between the two groups (p b 0.05).

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Fig. 6. Confocal microscopy images of MC3T3-E1 cells incubated with NDs and Alen-NDs (both have green fluorescence) at 3 and 12 h. F-actin (red) and nuclei (blue) were stained with Alexa Fluor 546 phalloidin and 4-6-diamidino-2-phenylindole (DAPI), respectively.

confocal microscopy images matched to the corresponding data in Fig. 5. In addition, MC3T3-E1 cells were cultured with Alen, NDs, and AlenNDs in an osteogenic medium and ALP activity was measured after 1, 5, and 7 days of culture. As shown in Fig. 7, both Alen and NDs increased ALP activity, compared to the control group (osteogenic medium only). The enhanced ALP activity by Alen was previously reported [32]. The positive effect of NDs on ALP activity was attributed to the preference of MC3T3-E1 cells for rough and hard surfaces, as many researchers have demonstrated using HAp, titanium, and bioactiveglass [38]. Note that the treatment of Alen-NDs led to a significant increase in ALP activity. ALP activity of the Alen-NDs group after 7 days was 2.2-fold higher compared to that of the Alen and 1.6-fold higher compared to that of the NDs. Therefore, this enhanced ALP activity was attributed to synergy of Alen and NDs. 3.5. Comparison of performance in therapy To demonstrate the superior properties of ND-based drug delivery system, PLGA was selected as a material for drug carriers. PLGA nanoparticles were prepared by nanoprecipitation method in a simple fluidic device and further conjugated with Alen for comparison with Alen-NDs.

Fig. 7. Alkaline phosphatase (ALP) activities of MC3T3-E1 cells after 1, 5, and 7 days of incubation with Alen, NDs, and Alen-NDs. Untreated cells served as the control (osteogenic medium only). Data are mean ± standard deviation (n = 3). ⁎Significant difference between the two groups (p b 0.05).

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Fig. 8. (a) Cellular uptake ratio of NDs, PLGA, Alen-NDs, and Alen-PLGA in MC3T3-E1 cells after 3 and 12 h incubations. (b) ALP activity of MC3T3-E1 cells after 1, 5, and 7 days of incubation with PLGA, Alen, and Alen-PLGA, where osteogenic medium only served as control. All the samples were dispersed in osteogenic medium before treatment. The data are presented as mean ± standard deviation (n = 3). ⁎Significant difference between the two groups (p b 0.05).

The average size of the Alen-PLGA nanoparticles was adjusted to 63.06 ± 9.50 nm similar to that (64.74 ± 20.76 nm) of Alen-NDs (Fig. S7). Fig. 8a and b show the cellular uptake behavior and ALP activity of MC3T3-E1 cells treated with each type of the samples, respectively. There was no significant difference in cellular uptake ratio among the NDs, PLGA, Alen-NDs, and Alen-PLGA groups at 3 h. In all cases, the amount of cellular uptake increased at 12 h. The cellular uptake ratio (13.24%) of NDs was significantly higher than that (8.75%) of PLGA nanoparticles. Alen conjugation led to an increase in the cellular uptake ratio and Alen-NDs had a higher cellular uptake ratio (20.21%) compared to Alen-PLGA (14.67%). Note that the NDs showed a similar cellular uptake ratio to Alen-PLGA. This result suggests that NDbased drug carriers can be more effectively uptaken by the MC3T3-E1 cells.

Fig. 8b shows ALP activity of MC3T3-E1 cells treated with PLGA, Alen, Alen-PLGA with respect to time. At 1 day, there was no significant difference in ALP activity among the samples. At 5 and 7 days, PLGA nanoparticles had the similar ALP activity to control (osteogenic medium). The slight increase in ALP activity of PLGA group over time is attributed to the presence of osteogenic medium. Alen-PLGA nanoparticles also had the similar ALP activity to Alen. This result revealed that the PLGA itself had no effect on ALP activity. As observed in Fig. 7, NDs itself enhanced ALP activity of MC3T3-E1 cells. ALP activity induced by NDs was 6.04 μmol/μg ∙ min at 7 days, which was greater than those of PLGA nanoparticles (1.29 μmol/μg ∙ min) and Alen only (4.16 μmol/μg ∙ min). Therefore, it can be concluded that NDs-based drug carrier had superior performances in terms of cellular uptake and ALP activity of MC3T3-E1 cells.

Fig. 9. In vivo non-invasive fluorescence images of (a) nude mice 12 h after an intravenous tail vein injection of Alen, NDs, and Alen-NDs. RITC solution in PBS served as the control, and the RITC dose was kept at 1.8 × 10−5 mol/kg mouse. (b) Ex vivo fluorescence images of excised organs (kidney, heart, spleen, lung, and liver).

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3.6. In vivo bone-targeted delivery Instead of FITC, RITC was conjugated to Alen and/or NDs for the in vivo study to avoid fluorescence interference [25]. To evaluate the potential in an animal study, RITC-conjugated Alen, NDs, and AlenNDs were injected into mice through the tail vein, and aqueous RITC solution was used as a control. The Alen-NDs administered through tail vein pass through heart, lungs, and heart again, and then distributed to the organs of the body (e.g., liver, kidney, and spleen), finally accumulated in bone. Fig. 9a shows non-invasive fluorescent images of the mice after 12 h post-injection. Most RITC molecules and NDs were observed in abdominal organs (e.g., kidneys and liver) [39]. In contrast, Alen and Alen-NDs were mainly observed in bone tissues, particularly those in the arms, feet, and ears. The high fluorescence intensity on the ears may have been due to their high capillary density and very low thickness of ear tissue under the limited penetration depth of the image station. To evaluate biodistribution, the organs were explanted from the mice (Fig. 9b). Strong fluorescence intensity was observed primarily in liver of the control group (aqueous RITC solution), as typically observed in other studies [40,41]. In the cases of NDs group, strong fluorescence intensities were mainly detected in the kidneys, heart, lungs, and liver. Note that the fluorescence intensity in the organs of the mice treated with Alen-NDs was significantly lower, compared to that of the Alen group, suggesting their high targeting efficiency to bone.

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These results confirmed that Alen-NDs were efficiently deposited in bone throughout the body. To confirm the targeting capabilities of Alen and Alen-NDs, a vertebra was excised from the treated animals. Fig. 10a and b show the vertebral images taken by Image Station and fluorescent microscopy, respectively. Vertebra in the Alen and Alen-NDs groups exhibited strong fluorescence compared to that of the control and ND groups. As shown in Fig. 10c, fluorescence intensity was quantitatively assessed for comparison. Animals treated with Alen exhibited very high level of fluorescence intensity, compared to that of the control. Alen-NDs exhibited 8.8-fold higher fluorescence intensity than that of the NDs. In addition, animals treated with Alen-NDs had a significantly higher fluorescence intensity even compared to that of Alen. In in vitro study, Alen showed the highest binding property to HAp (Fig. 3). In contrast, the in vivo result revealed the small difference in fluorescence intensity between Alen and Alen-NDs. This discrepancy between in vitro and in vivo results might be attributed to the large differences in size of Alen (approximately b10 nm) and Alen-NDs (approximately 60 nm). Alen molecules could be bound to the HAp powder at a higher density than Alen-NDs, resulting in the higher in vitro binding ratio of Alen. Despite the discrepancy, the in vivo result might be more relevant for practical applications. These results clearly confirm the superior targeting efficiency of Alen-NDs to bone tissues, suggesting promising potential for bone targeted delivery.

Fig. 10. (a) Ex vivo fluorescence images, (b) fluorescent microscopic images, and (c) a plot of relative fluorescence intensity of excised vertebra 12 h after an intravenous tail vein injection of Alen, NDs, and Alen-NDs. RITC solution in PBS served as the control, and the RITC dose was kept at 1.8 × 10−5 mol/kg mouse. Data are mean ± standard deviation (n = 3). ⁎Significant difference between the two groups (p b 0.05).

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4. Conclusions We successfully fabricated bone-targeted drug carriers based on NDs and demonstrated their superior performances in terms of HAp affinity, specific uptake for MC3T3-E1 cells, positive synergistic effect for ALP activity, and specific accumulation to bone tissue. Among them, the enhanced synergistic ALP activity induced by NDs and Alen makes NDs attractive materials for osteoporosis treatments. We believe that hard NDs accumulated in bone tissue can help rapidly increase in the mechanical strength of bone by being incorporated into bone tissue. Therefore, our next goal will be focused on therapeutic effect of NDs and Alen-NDs in an osteoporotic mouse model by evaluating mineral content and mechanical strength of bone tissue. Acknowledgements This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF2014R1A1A1005826 and NRF-2015R1A4A1042350) and a grant from the Korea Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (project no. HI13C1634). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jconrel.2016.04.025. References [1] C.S. Lee, W. Park, S.J. Park, K. Na, Endolysosomal environment-responsive photodynamic nanocarrier to enhance cytosolic drug delivery via photosensitizer-mediated membrane disruption, Biomaterials 34 (2013) 9227–9236. [2] S. Ganta, H. Devalapally, A. Shahiwala, M. Amiji, A review of stimuli-responsive nanocarriers for drug and gene delivery, J. Control. Release 126 (2008) 187–204. [3] X.L. Luo, A. Morrin, A.J. Killard, M.R. Smyth, Application of nanoparticles in electrochemical sensors and biosensors, Electroanalysis 18 (2006) 319–326. [4] H. Yoo, S.K. Moon, T. Hwang, Y.S. Kim, J.H. Kim, S.W. Choi, J.H. Kim, Multifunctional magnetic nanoparticles modified with polyethylenimine and folic acid for biomedical theranostics, Langmuir 29 (2013) 5962–5967. [5] A.J. Coughlin, J.S. Ananta, N. Deng, I.V. Larina, P. Decuzzi, J.L. West, Gadoliniumconjugated gold nanoshells for multimodal diagnostic imaging and photothermal cancer therapy, Small 10 (2014) 556–565. [6] Q. Zhang, V.N. Mochalin, I. Neitzel, I.Y. Knoke, J. Han, C.A. Klug, J.G. Zhou, P.I. Lelkes, Y. Gogotsi, Fluorescent PLLA-nanodiamond composites for bone tissue engineering, Biomaterials 32 (2011) 87–94. [7] B.T. Zhang, X. Zheng, H.F. Li, J.M. Lin, Application of carbon-based nanomaterials in sample preparation: a review, Anal. Chim. Acta 784 (2013) 1–17. [8] V.N. Mochalin, O. Shenderova, D. Ho, Y. Gogotsi, The properties and applications of nanodiamonds, Nat. Nanotechnol. 7 (2011) 11–23. [9] E.K. Chow, X.Q. Zhang, M. Chen, R. Lam, E. Robinson, H. Huang, D. Schaffer, E. Osawa, A. Goga, D. Ho, Nanodiamond therapeutic delivery agents mediate enhanced chemoresistant tumor treatment, Sci. Transl. Med. 3 (2011) (73ra21). [10] B. Guan, F. Zou, J. Zhi, Nanodiamond as the pH-responsive vehicle for an anticancer drug, Small 6 (2010) 1514–1519. [11] M.W. Orme, V.M. Labroo, Synthesis of β-estradiol-3-benzoate-17-(succinyl-12A-tetracycline): a potential bone-seeking estrogen, Bioorg. Med. Chem. Lett. 4 (1994) 1375–1380. [12] S. Kasugai, R. Fujisawa, Y. Waki, K. Miyamoto, K. Ohya, Selective drug delivery system to bone: small peptide (Asp)6 conjugation, J. Bone Miner. Res. 15 (2000) 936–943. [13] K. Yokogawa, K. Miya, T. Sekido, Y. Higashi, M. Nomura, R. Fujisawa, K. Morito, Y. Masamune, Y. Waki, S. Kasugai, K. Miyamoto, Selective delivery of estradiol to bone by aspartic acid oligopeptide and its effects on ovariectomized mice, Endocrinology 142 (2001) 1228–1233. [14] D. Wang, S. Miller, M. Sima, P. Kopecková, J. Kopecek, Synthesis and evaluation of water-soluble polymeric bone-targeted drug delivery systems, Bioconjug. Chem. 14 (2003) 853–859.

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