Aspartic acid-based modified PLGA–PEG nanoparticles for bone targeting: In vitro and in vivo evaluation

Acta Biomaterialia 10 (2014) 4583–4596

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Aspartic acid-based modified PLGA–PEG nanoparticles for bone targeting: In vitro and in vivo evaluation Yin-Chih Fu a,b,c, Tzu-Fun Fu d, Hung-Jen Wang e, Che-Wei Lin a,e, Gang-Hui Lee d, Shun-Cheng Wu a, Chih-Kuang Wang a,e,⇑ a

Orthopaedic Research Center, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Department of Orthopaedics, College of Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan d Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan e Department of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan b c

a r t i c l e

i n f o

Article history: Received 7 April 2014 Received in revised form 5 July 2014 Accepted 14 July 2014 Available online 19 July 2014 Keywords: Bone targeting Aspartic acid Nanoparticle PLGA–PEG Drugs delivery

a b s t r a c t Nanoparticles (NP) that target bone tissue were developed using PLGA–PEG (poly(lactic-co-glycolic acid)–polyethylene glycol) diblock copolymers and bone-targeting moieties based on aspartic acid, (Asp)n(1,3). These NP are expected to enable the transport of hydrophobic drugs. The molecular structures were examined by 1H NMR or identified using mass spectrometry and Fourier transform infrared (FT-IR) spectra. The NP were prepared using the water miscible solvent displacement method, and their size characteristics were evaluated using transmission electron microscopy (TEM) and dynamic light scattering. The bone targeting potential of the NP was evaluated in vitro using hydroxyapatite affinity assays and in vivo using fluorescent imaging in zebrafish and rats. It was confirmed that the average particle size of the NP was <200 nm and that the dendritic Asp3 moiety of the PLGA–PEG–Asp3 NP exhibited the best apatite mineral binding ability. Preliminary findings in vivo bone affinity assays in zebrafish and rats indicated that the PLGA–PEG-ASP3 NP may display increased bone-targeting efficiency compared with other PLGA–PEG-based NP that lack a dendritic Asp3 moiety. These NP may act as a delivery system for hydrophobic drugs, warranting further evaluation of the treatment of bone disease. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Non-union fracture, osteonecrosis (ON) and osteoporosis (OP) are common bone disorders that cause severe disability to the patient and a heavy socioeconomic burden on families and society [1]. Bone loss can lead to OP, which is associated with increased fracture risk, decreased bone strength, diminished quality of life and increased mortality [1,2]. Optimizing treatment efficacy will help to improve health care and decrease treatment costs. Treatment options include exercise, calcium supplementation, postmenopausal estrogen therapy, calcitonin therapy, and the administration of bisphosphonates (BP), parathyroid hormone (PTH) or calcitriol. Current treatments for OP mainly use drugs to suppress bone resorption, and the treatment of fracture and ON typically involves surgery. To improve the efficacy of treatments

⇑ Corresponding author at: Department of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan. Tel.: +886 7 3121101x2677; fax: +886 7 3125339. E-mail address: [email protected] (C.-K. Wang). http://dx.doi.org/10.1016/j.actbio.2014.07.015 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

for OP and non-union fracture, agents with an enhancing effect on bone formation are preferred [3–7]. Chemical agents often result in side effects and have low efficacy. For example, PTH is the only bone anabolic agent clinically approved to stimulate bone formation in severe OP [8]. However, PTH treatment is limited to a 2-year period because of increasing bone resorption over bone formation and the potential risk of developing osteosarcoma [9,10]. In addition, statins and isoflavone derivatives have also been reported to stimulate osteogenic differentiation, but these derivatives are not hydrophilic [11]. Therefore, these poorly water-soluble drugs cannot be administered by intravenous (IV) injection, and effective drug concentrations are attained using oral medicine. Because musculoskeletal diseases lack targeting drug delivery systems, drugs cannot be delivered to a specific region. In addition, high systemic doses of the therapeutic agent must often be administered, which can lead to significant adverse systemic and side effects. Bone disease states cause local inflammation and/or result in the exposure of hydroxyapatite (HAp) to blood [12]. The exposure of HAp to blood introduces unique targeting possibilities. Various tetracyclines, BP, acidic oligopeptides (AO), chelating compounds

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and salivary proteins have all been employed to target bone diseases. These compounds bind to the inorganic HAp component of bone and exhibit unique binding affinities, which may affect the disease states for which they are most appropriate [12,13]. For example, Wang et al. [14] confirmed that BP are less affected by crystallinity, while aspartic acid (Asp) prefers more crystalline HAp. Miller et al. [15] demonstrated that tetracycline is deposited preferentially onto surfaces that are growing or that display low crystallinity. Furthermore, osteoclast targeting can be achieved with AO, which prefer absorbing surfaces, while osteoblast targeting should employ tetracycline, which binds preferentially to growing bone surfaces [12]. In addition, AO binds to HAp faster than BP does [16]. This may be due to the larger binding area of AO [12]. Sekido et al. [17] compared the binding rates of (Asp)2–10 to HAp. Increases in Asp chain length resulted in enhanced binding rates to hexapeptides; the rate of binding then plateaued, and further increases in the chain length produced no effect. Accordingly, it is of interest to develop a novel strategy to increase bone formation for the prevention and treatment of OP. Moreover, such a strategy might also play a role in the treatment of the non-union of fractures. Controlled drug delivery can improve efficacy, reduce toxicity and improve patient compliance and convenience [18]. An efficient carrier can transport a useful chemical agent to bone tissue; the chemical agent can then promote complete bone growth. Biodegradable poly(hydroxyl acid) compounds, such as copolymers of poly(lactic acid) (PLA) and poly(lactic-coglycolic acid (PLGA), are being used extensively in biomedical applications because of their biocompatibility, their ability to encapsulate various drug molecules, and their sustained release properties. PLGA NP can release their contents over a wide time scale [19,20]. Therefore, new bone-targeting drug delivery systems have been developed using PLGA-based NP. Choi and Kim [21] designed PLGA–PEG (PLGA-graft-polyethylene glycol) block copolymers intermixed with alendronate (ALN)-functionalized PLGA to create surface-modified NP, and ALN was used as the bone-targeting moiety. Their results revealed decreasing adsorption with decreasing concentrations of ALN at the HAp surface; in addition, increases in the PEG mole ratio (550, 750 and 2000 Mn) resulted in decreased adsorption, apparently because of shielding of the ALN moieties by longer PEG chains. Pignatello et al. [22] also obtained a PLGA derivative via conjugation with ALN. With this derivative, copolymer NP were produced and loaded with doxorubicin as a model anticancer drug. It was demonstrated both in vitro and in vivo that the encapsulation of the drug did not affect its anticancer activity. Unlike the other linear aspartic acid oligopeptides reported in the literature, which are 4–10 amino acids long [17,23,24], the present authors approached the rational design of a NP delivery system for hydrophobic drugs using dendritic Asp3 moieties to form PLGA–PEG–Asp3 copolymers that specifically target bone tissue (Fig. 1a, c, d). In accordance with the previously reported method developed in the present authors’ laboratory [25], this system depends on PEG–PLGA block copolymers and bone targeting moieties that contain (Asp)n(1,3), thus facilitating the delivery of therapeutic agents to bone tissue. In addition, the system combines different ratios of fluorescent probe copolymers of fluorescein isothiocyanate (FITC)–PLGA–PEG (Fig. 1b), conferring the bone-targeting NP with optical image tracking capabilities. PLGA–PEG– COOH (PLGA–carboxyl polyethylene glycol) and PLGA–PEG–OMe (PLGA–monomethoxy polyethylene glycol) were also synthesized as control groups. To evaluate the in vitro affinity of HAp powder and its ability to be endocytosed by the D1 bone marrow stem cell line, a bone targeting in vivo feasibility assessment was performed using fluorescent imaging experiments in zebrafish and Sprague–Dawley (SD) rats. Every synthesis step in the creation of the copolymers was

systematically identified, and the NP properties, such as the average size distribution, morphology and cell toxicity, were characterized. This bone delivery system could be the foundation for hydrophobic-drug-based therapies that draw on basic science in the bone tissue field. 2. Materials and methods 2.1. Materials L-Aspartic acid (HO2CCH2(NH2)CO2H), N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride, nuclear magnetic resonance (NMR) d-solvents, tetramethylsilane (TMS), calcium hydride and PLGA (50/50, Mw 50,000–75,000, no. 430447) were purchased from Sigma-Aldrich Co. (USA), and Boc–Asp–OH (tert-butyl carbamates–Asp–OH) was purchased from FlukaÒ (Sigma-Aldrich Co. USA). H2N–PEG–COOH (Mw 3400) was purchased from Laysan Bio., Inc. (USA); N,N0 -dicyclohexylcarbodiimide (DCC) and N-hydroxysulfosuccinimide (Sulfo-NHS) were purchased from Acros Organics (USA); and trifluoroacetic acid, hydrochloric acid and triethylamine (TEA) were purchased from Riedel-de HaenÒ (Sigma-Aldrich Co., USA). Sodium hydroxide and magnesium sulfate anhydrous powder were purchased from Showa PK Co. (Japan). All other solvents were purchased from TEDIA (USA) or J.T. Baker (USA).

2.2. Synthesis of the Asp1–(OMe)2 moiety and PLGA–PEG–Asp1 copolymers The copolymers of PLGA–PEG–Asp1 were prepared from PLGA– PEG, and the end group was modified with aspartic acid (Asp1). The Asp1–(OMe)2 moiety and PLGA–PEG–Asp1 copolymer synthesis mechanism are illustrated in Scheme 1. First, the two carboxyl groups of aspartic acid were methylated into an Asp1–(OMe)2 moiety, as described below, using 100 ml of methanol and 16 ml (0.225 mmol) of thionylchloride (SOCl2) in a 250-ml two-necked flask immersed in an ice bath for 30 min, followed by the slow addition of 15 g (0.112 mmol) of aspartic acid. This solution was heated and refluxed for 5 h, then dried using a vacuum system to create a powder. The powder was dissolved in 20 ml of methanol, and the ester was used to precipitate the Asp1–(OMe)2 in Scheme 1a. To collect the precipitate, the material was dried in a vacuum system to yield a white powder. The intermediate product of NH2–PEG–Asp1 in Scheme 1c was prepared to obtain the PLGA–PEG–Asp1 copolymers, which were prepared as follows. First, 100 mg (0.029 mmol) of NH2–PEG– COOH and 8.45 mg (0.044 mmol) of DCC were mixed in a 25-ml flask (A) and mounted in a vacuum system. Then, flask A was evacuated and purged using pure nitrogen. Next, 5 ml of dry dimethylformamide (DMF) was used as a solvent to dissolve the NH2–PEG–COOH and DCC, then left to react for 1 h. The mixture solution in flask A was streamed into flask B, which contained 5.07 mg (0.044 mmol) of Sulfo-NHS and 14.25 mg (0.088 mmol) of Asp1–(OMe)2, which had also been pre-treated by vacuum. Then, 0.1 ml of TEA was added to flask B, left to react for 6 h and purged using pure nitrogen. A glassy filter was used to remove the precipitate, and the solution product was placed in a dialysis bag (MWCO 1000) and placed in methanol to exchange the DMF; the methanol solvent was changed every 2 h for 12 h. Then, the dialysis solution was exchanged for deionized water, and the product was freezedried to create the NH2–PEG–Asp1–(OMe)2 of Scheme 1b. The methyl groups were removed from the NH2–PEG–Asp1–(OMe)2 of Scheme 1c, creating NH2–PEG–Asp1. The NH2–PEG–Asp1–(OMe)2 was dissolved in 15 ml of methanol and reacted with 0.5 M NaOH under a pH of 12 for 4 h. Then, a small amount of 1 M HCl was

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Fig. 1. Rational design (d) of bone targeting nanoparticles (NP) composed of (a) aspartic acid functionalized PLGA–PEG-based copolymers, which can carry (b) a fluorescent tracer (FITC) conjugated with PLGA–PEG-based copolymers and (c) therapeutic agents (hydrophobic drug).

Scheme 1. The synthesis mechanism and steps used to make (a) the Asp1–(OMe)2 moiety and (b)–(d) PLGA–PEG–Asp1 copolymers.

added to the solution to maintain a pH of 7. Finally, the solution product was placed in a dialysis bag (MWCO 1000) and placed in deionized water; the deionized water was changed every 2 h for 12 h, and the pale yellow product was freeze-dried to produce the NH2–PEG–Asp1 of Scheme 1c. The final amphiphilic copolymer (4 of Scheme 1d) of PLGA– PEG–Asp1 was produced as follows: 100 mg (0.029 mmol) of NH2–PEG–Asp1 was vacuum-dried by rotary evaporation, dissolved

in 5 ml of dry DMF in flask A and subsequently re-added to 0.1 ml of TEA for 4 h under nitrogen. Next, 300 mg (0.025 mmol) of PLGA (50/50) and 8 mg (0.039 mmol) of DCC were also dried by vacuum and added to 15 ml of dry DMF in flask B to react for 4 h under nitrogen. The flask A solution of NH2–PEG–Asp1 was mixed with the flask B solution of PLGA (50/50) and DCC, and the mixture was left to react overnight. A glassy filter was used to remove the precipitate, and the solution product was placed in a dialysis

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bag (MWCO 13,000–14,000) and placed in dry DMF to exchange; the dry DMF solvent was changed every 2 h for 12 h. The final stage of the process was to allow the dialysis to occur in deionized water; the deionized water was changed every 2 h for 12 h, and the white product was freeze-dried to create the PLGA–PEG–Asp1 in Scheme 1d. 2.3. Synthesis of the Asp3–(OMe)4 moiety and PLGA–PEG–Asp3 copolymers The copolymers of PLGA–PEG–Asp3 were also prepared from PLGA–PEG, and the end group was modified with a dendritic trimer of aspartic acid oligopeptide (Asp3). The Asp3–(OMe)4 moiety and PLGA–PEG–Asp3 copolymer synthesis mechanism and steps are shown in Scheme 2. First, the Boc-protected amine groups of aspartic acid (Boc–Asp–OH) were reacted with Asp1–(OMe)2 to form an Asp3–(OMe)4 moiety, as follows. First, 1.475 g (6.33 mmol) Boc– Asp–OH and 2.5 g (12.66 mmol) Asp1–(OMe)2 were placed in the 100-ml flask A and dissolved in 10 ml chloroform, followed by the addition of 1.77 ml (6.33 mmol) TEA under an ice bath for 30 min. Next, 10 ml of tetrahydrofuran was used as a solvent to dissolve 3.2 g (15.1 mmol) DCC in the 100-ml flask B, and the resulting solution was injected into flask A to react for 24 h at 5 °C. A glassy filter was used to remove the precipitate, and the product was freeze-dried to create the Boc–Asp3–(OMe)4 in Scheme 2a. The Boc group was removed from Boc–Asp3–(OMe)4, yielding the Asp3–(OMe)4 in Scheme 2b. Then, 1 g Boc–Asp3– (OMe)4 was dissolved in 10 ml dichloromethane (DCM) within the 100-ml flask and added to 4 ml of TEA at room temperature for 1 h. Then, the product was vacuum-dried by rotary evaporation and subsequently re-dissolved in DCM; this process was repeated three times. Finally, the oily substance was extracted using ester and vacuum-dried to produce Asp3–(OMe)4 in the form of a pale yellow powder. Similarly, the intermediate product NH2–PEG–Asp3 in Scheme 2c and d was prepared to produce the PLGA–PEG–Asp3 copolymers; this product was synthesized as follows. First, 100 mg (0.029 mmol) NH2–PEG–COOH and 8.45 mg (0.044 mmol) DCC were mixed in the 25-ml flask A and mounted into a vacuum system. Then, flask A was evacuated and purged using pure nitrogen. Next, 5 ml of dry DMF was used as a solvent to dissolve the NH2–PEG–COOH and DCC, and the solution was left to react for 1 h. The mixture solution in flask A was streamed into flask B, which contained 5.07 mg (0.044 mmol) Sulfo-NHS and 36.98 mg (0.088 mmol) Asp3–(OMe)4, which had also been pre-treated in a vacuum. Then, 0.1 ml of TEA was added to flask B, allowed to react for 6 h and purged using pure nitrogen. A glassy filter was used to remove the precipitate, and the solution product was placed in a dialysis bag (MWCO 1000), which was immersed in methanol to exchange the DMF; the methanol solvent was changed every 2 h for 12 h. Then, the dialysis solution was exchanged for deionized water, and the product was freeze-dried to create the NH2–PEG– Asp3–(OMe)4 in Scheme 2c. The methyl groups were removed from the NH2–PEG–Asp3–(OMe)4 in Scheme 2d, creating NH2–PEG–Asp3. The NH2–PEG–Asp3–(OMe)4 was dissolved in 15 ml of methanol and reacted with 0.5 M NaOH under a pH of 12 for 4 h. Then, a small amount of 1 M HCl was added to the solution to maintain a pH of 7. Finally, the solution product was placed in a dialysis bag (MWCO 1000), which was immersed in deionized water; the deionized water was changed every 2 h for 12 h, and the pale yellow product was freeze-dried to create the NH2–PEG–Asp3 of Scheme 2d. According to the previous method, the final amphiphilic copolymer (9 in Scheme 2e) of PLGA–PEG–Asp3 was produced as follows. First, 100 mg (0.026 mmol) of NH2–PEG–Asp3 was vacuum-dried by rotary evaporation, dissolved in 5 ml of dry DMF in flask A and subsequently re-added to 0.1 ml of TEA for 4 h under nitrogen.

Next, 300 mg (0.025 mmol) PLGA (50/50) and 8 mg (0.039 mmol) DCC were also dried by vacuum and added to 15 ml of dry DMF in flask B to react for 4 h under nitrogen. The flask A solution of NH2–PEG–Asp1 was mixed with the flask B solution of PLGA (50/50) and DCC, and the mixture was left to react overnight. A glassy filter was used to remove the precipitate, and the solution product was placed in a dialysis bag (MWCO 13,000–14,000) and then in dry DMF for exchange; the dry DMF solvent was changed every 2 h for 12 h. The final stage of the process was to allow the dialysis to occur in deionized water; the deionized water was changed every 2 h for 12 h, and the white product was freeze-dried to create the PLGA–PEG–Asp3 in Scheme 2e. 2.4. Synthesis of other control copolymers and fluorescent-probe copolymers 2.4.1. Synthesis of the PLGA–PEG–OMe and PLGA–PEG–COOH copolymers In brief, 300 mg (0.025 mmol) PLGA (50/50) was activated with 8 mg (0.039 mmol) DCC in 3 ml of anhydrous DMF under an inert atmosphere for 4 h (a PLGA:DCC mole ratio 1:1.5). In a separate flask, 100 mg (0.029 mmol) NH2–PEG–OMe or NH2–PEG–COOH was dissolved in 2 ml anhydrous DMF with 0.1 ml TEA for 4 h and then added to the activated PLGA solution in a drop-wise manner (PLGA:NH2–PEG–OMe (or NH2–PEG–COOH); mole ratio 1:1.2). The reaction mixture was stirred under an inert nitrogen atmosphere overnight. The solution was dialyzed (MWCO 1000 Da) against distilled water for 12 h to remove the unreacted NH2–PEG–OMe or NH2–PEG–COOH and then freeze-dried. 2.4.2. Synthesis of the FITC–PLGA–PEG copolymers To further investigate the cellular uptake of these NP, an mPEG– PLGA–FITC copolymer with a fluorescent moiety was synthesized as described in Ref. [26], with minor modifications. In brief, 300 mg (0.025 mmol) PLGA (50/50) was activated with 8 mg (0.039 mmol) DCC in 3 ml anhydrous DMF under an inert atmosphere for 4 h (PLGA:DCC mole ratio of 1:1.5). In a separate flask, 100 mg (0.029 mmol) NH2–PEG–OMe was dissolved in 2 ml anhydrous DMF with 0.1 ml TEA for 4 h and then added to the activated PLGA solution in a drop-wise manner (PLGA:NH2–PEG–OMe mole ratio of 1:1.2). The reaction mixture was stirred under an inert nitrogen atmosphere overnight. The solution was dialyzed (MWCO 1000 Da) against distilled water for 12 h to remove the unreacted NH2–PEG–OMe and then freeze-dried. Subsequently, 20 mg (0.05 mmol) FITC was conjugated to 1.5 g (0.1 mmol) mPEG–PLGA in 30 ml DMSO at 90 °C for 2 h in the dark. The resulting solution was dialyzed against distilled water for 3 days to remove the DMSO and unreacted FITC; the solution was then lyophilized. 2.5. Characterization of all synthesis copolymers These copolymers were identified using NMR, mass spectrometry (MS) and IR spectroscopy. 1H-NMR spectra were recorded on a Varian Gemini-200 or a Varian Gemini-600 spectrometer using TMS as an internal standard. The chemical shifts are given in d (ppm), and the coupling constants are given in hertz. Small molecular weights (<3000 Da) were detected by electrospray ionization (ESI)-MS on a JMS-HX 100 mass spectrometer. IR spectra were obtained using a Perkin-Elmer System 2000 FT-IR spectrophotometer. 2.6. Formulation and characterization of the bone-targeting nanoparticles To prepare the NP colloids, 25 mg of each copolymer was dissolved completely in 1 ml of DMF that are miscible with water.

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Scheme 2. The synthesis mechanism and steps used to make (a) the Asp3–(OMe)4 moiety and (b)–(e) PLGA–PEG–Asp3 copolymers.

NP were formed by adding the polymer solution to 2 ml of distilled water using the solvent displacement method. The resulting NP suspensions were allowed to stir uncovered for 10 min at room temperature. The resulting NP solution was dialyzed against distilled water for 2 days to remove the DMF [27]. The particle size distributions and zeta potential of these NP solutions were measured using a Zetasizer 1000 zeta potential instrument (Zetasizer 3000-HSA & Mastersizer M-2000 P-III, Malvern). The morphology and size of the NP were observed using transmission electron microscopy (TEM; JEM-2100, JEOL Ltd., Japan). A drop of NP suspension was placed on a carbon-coated copper grid, and the grid was dried at room temperature. The copper grid was stained for

2 min in a 2.0% phosphotungstic acid solution (pH 7.0). The choice of negative stain of phosphotungstic acid in electron microscopy can be very effective, because it scatters electrons well and also adsorbs well into biological matter [28]. 2.7. Bone mineral binding ability in vitro The adhesion between bone-like substrates and bone-targeting NP was demonstrated in vitro. The HAp powder binding capacity was evaluated using a method similar to that described in a previous report [29]. A brief description is as follows: the hydrophobic fluorescent dye Prodan (2.5 mg) and 25 mg of the copolymers were

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first dissolved in 1 ml DMF. Then, a method similar to that described above to prepare the NP colloid was used. Subsequently, the NP colloids containing Prodan from four types of copolymers were diluted to 10 ml as stock colloids; their initial fluorescence emission intensities were measured using a fluorescence spectrometer (Varian, Casry Eclipse) with emission at 430 nm. Then, 20 mg of HAp powder (CAPTALÒS, Plasma Biotal Limited, UK) with Ca10(PO4)6(OH)2 > 97.5%, an average particle size of 5 lm and an average crystallinity of 89.9% was added to 2 ml of each of the NP colloids containing Prodan. These suspensions were shaken at 1000 rpm for 1 h to allow the adsorption of the Prodan/NP onto the HAp powders using a Vortex mixer (RVM-102, REXMED Industries Co., Ltd.). The amount of Prodan/NP adsorbed was determined by measuring the difference between the fluorescence emission intensity of the initial stock solution and that of the supernatant after adsorption: HAp adsorption affinity ¼

Iinitial intensity  Isupernatant intensity after HAp adsorption  100% Iinitial intensity

2.8. Cell viability assay

(Axiovert 200, Carl Zeiss, Inc.). The images were recorded separately in each fluorescence channel and merged afterwards. 2.10. Evaluation of the bone-targeting nanoparticles in zebrafish in vivo 2.10.1. Blood vessel injection in zebrafish larvae Zebrafish larvae at the 7th day post fertilization (dpf) were anesthetized with tricaine (A5040, Sigma-Aldrich) and placed on a soft agar plate. The larvae were injected with 4.6 nl of a FITC-labeled hybrid NP (PLGA–PEG–Asp3/mPEG–PLGA–FITC or PLGA–PEG–OMe/mPEG–PLGA–FITC at a weight ratio of 8/2) colloid containing 0.125% Texas Red-dextran (D-1830, Molecular Probes) in the common cardinal vein and moved to fresh embryo water immediately after injection for recovery. The injected larvae were observed under a fluorescence dissecting microscope (Leica Microsystems, Germany) at 0.5 and 24 h post injection (hpi). 2.10.2. In vivo bone staining in zebrafish larvae Calcein staining for larval bone was performed following the protocol described previously [30]. In brief, the zebrafish larvae were immersed in a 0.2% calcein solution in water for 10 min, briefly rinsed in fresh water to remove superfluous dye and observed immediately under a fluorescence dissecting microscope.

Changes in the viability of the D1 cells (BALB/c mouse bone marrow mesenchymal stem cells (BMSC)) were quantitatively assessed using a tetrazolium compound (MTS; Sigma, USA) for one 24- or 72-h culture period. This tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium inner salt (MTS), produces a watersoluble formazan product that has an absorbance maximum at 490 nm in phosphate-buffered saline in the presence of phenazine methosulfate (PMS). The amount of colored product formed is proportional to the number of cells and their time of incubation with MTS/PMS. To evaluate the cell viability while in direct contact with the cells, a concentration of 5  105 cells per well was seeded on a 96-well cell culture plate and cultured at 37 °C for 2 days in a humidified incubator containing 5% CO2. Four types of NP colloids (20 ng ml1) were prepared by adding the culture medium that was previously incubated at 37 °C overnight and sterilized by UV light. Culture plates without any samples were used as the normal control. After 24 h of cultivation, 10 ll of an MTS solution (5 mg ml1 in PBS) was added to each well and incubated for another 4 h (37 °C). The optical density of the water-soluble formazan produced in the solution was measured using an ELISA reader (SLT, Crailheim, Germany). Data were collected and averaged from five different wells per condition. The data were statistically analyzed, and the results are expressed as the mean ± standard deviation.

The bone-targeting capacities of two types of NP (PLGA–PEG– Asp3 and PLGA–PEG–OMe) encapsulated with near-infrared (NIR) lipophilicity fluorescence dye (IR-780 iodide; Aldrich, USA) following the protocol described in Section 2.7 were evaluated in vivo using SD rats (purchased from BioLASCo Taiwan Co., Ltd.). The protocol approved by the national guidelines for the care and use of laboratory animals was observed, and the study was approved by the Animal Experimental Ethics Committee of KMU. Three 6-month-old female SD rats were divided into two groups, one with the NP of the PLGA–PEG–OMe control and the trial groups, and one with the bone targeting NP of PLGA–PEG–Asp3. For each experiment, three animals per group were used, and 1 ml of the NP colloids was injected into the tail vein of each rat; after the injection, all the animals had free access to food and water. After 48 h, the animals were sacrificed, and the major organs (heart, liver, spleen, lung, kidney, bilateral femur/tibia of hind limb bone, and vertebra) were collected. The fluorescence signals (430–820 nm) in these organs from three rats in each group were detected using in vivo imaging systems (IVIS 200 Imaging System, Caliper Life Science, Alameda, CA). All the organs of the six rats in each group were quantified using IVIS 200 software.

2.9. Endocytosis

2.12. Statistical analysis

To further investigate the cellular uptake ability of the NP of PLGA–PEG–Asp3, an mPEG–PLGA–FITC copolymer was synthesized as described previously in Section 2.4.2. Both PLGA–PEG–Asp3 and mPEG–PLGA–FITC (at a weight ratio of 8/2) were used to form FITC-labeled hybrid NP in the same manner previously described to prepare the NP. D1 cells were also seeded onto a borosilicate chambered cover glass (Nunc, USA) at a density of 2  105 cells per well at 37 °C for 24 h. Then, the spent growth medium was replaced with 2 ml of the FITC-labeled hybrid NP solution in serum-free media. After different time intervals (10, 15, 20 min) of incubation at 37 °C, the cells were washed three times with PBS, followed by fixation and counterstaining with 40 ,6-diamidino-2-phenylindole (DAPI). The cells were then observed and imaged under 60 magnification using a confocal microscopy unit

Mean ± SD values were used for the expression of data, unless noted otherwise. Statistical analyses of the data were performed using Student’s t test. Differences characterized by p < 0.05 were considered statistically significant.

2.11. Evaluation of the bone-targeting nanoparticles in rats in vivo

3. Results and discussion 3.1. Synthesis and characterization of copolymers Four types of copolymers (PLGA–PEG–OMe, PLGA–PEG–COOH, PLGA–PEG–Asp and PLGA–PEG–Asp3) were successfully synthesized. The dendritic Asp3 moiety for the bone-targeting copolymer of PLGA–PEG–Asp3 was synthesized by direct conjugation of PLGA– COOH with H2N–PEG–Asp3 by amide linkage using the stepwise

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method presented in Scheme 1a and Scheme 2a–e, which included six steps. However, the bone-targeting moiety trimer aspartic acid (Asp3–(OMe)4) was synthesized first, and then the Asp3–(OMe)4 was reacted with NH2–PEG–COOH to form H2N–PEG–Asp3– (OMe)4. Therefore, the as-received copolymer of H2N–PEG–Asp3 was obtained after removing OMe from H2N–PEG–Asp3–(OMe)4. To characterize the Asp3–(OMe)4 moiety, the product was evaluated by 1H NMR spectroscopy and ESI-MS, as shown in Fig. 2a and b. The 1H NHR spectra reveal triplet peaks at 3.61–3.82 ppm that correspond to OCH3; the peak at 3.12 ppm represents CH2, and the peak at 4.41 ppm represents CH. The ESI-MS spectrum revealed a molecular weight of 420.20 Da, indicating that the synthesis of the Asp3–(OMe)4 moiety was successful. The 1H-NMR signals of the OCH3 in Asp3–(OMe)4 and of the ether linkages in PEG at 3.64 ppm overlap. The other 1H-NMR signals of Asp3 are much smaller than those of PEG; therefore, the 1H-NMR signals of Asp3 are not visible. The chemical structure of H2N–PEG–Asp3–(OMe)4 was thus further confirmed using FT-IR spectroscopy (Fig. 2c). Following methylation, the carboxylic groups of the aspartic acid side chains are methylated, i.e. the O@CAOH groups are converted to O@CAOCH3; the FT-IR spectrum of Asp3–(OMe)4 revealed a peak at 1729 cm1 and a second peak at 1612 cm1, indicating the presence of O@C and CAO bonds, respectively. The FT-IR results from H2N–PEG–COOH indicated antisymmetric stretching of the ether group (1095 cm1) in the spectrum. However, the NH2–PEG–COOH reacted with Asp3–(OMe)4, and peaks at 1729 cm1 and 1612 cm1 were also observed, indicating the presence of ester and amide bonds, respectively. However, the NH2–PEG–COOH was retained in the product, suggesting the formation of a NH2–PEG–Asp3– (OMe)4 copolymer. The FT-IR results indicated successful conjugation of H2N–PEG–COOH and Asp3, as expected. However, the segment copolymer of H2N–PEG–Asp3 can directly conjugate to PLGA–COOH through an amide linkage to form the bone-targeting copolymer PLGA–PEG–Asp3. 1H NMR spectra verified the PLGA–PEG–Asp3 structure (Fig. 3), showing a signal peak at 1.56 ppm representing PLA methyl protons (PLA CH3), a peak at 4.66–4.81 ppm representing PGA methylene protons (CH2), and a peak at 3.64 ppm representing PEG ether linkages. However, the 1H-NMR signals of Asp3 were also much smaller than those of PLGA and PEG and were not visible. Therefore, the 1H-NMR spectra confirmed that H2N–PEG–Asp3 was incorporated into the PLGA chain. The other three products (PLGA–PEG–OMe, PLGA–PEG–COOH and PLGA–PEG–Asp1) of each step are verified in the supporting information (Fig. S1–4). 3.2. Characterization of the four types of nanoparticles The basic structure of the bone targeting copolymers used to form the NP is shown in Fig. 1. These functional NP were designed to contain head groups on their surfaces, with the reduced immunogenic moieties of PEG conjugated to the biodegradable hydrophobic PLGA portion. The dendritic trimers in the aspartic acid oligopeptide (Asp3) head groups of the NP were created for bone targeting, and the size of the NP determined their cellular uptake. A polymer larger than 400 nm is restrictive [31], while a size of 100–200 nm can offer better cellular uptake [32]. To prepare these NP, 25 mg of the copolymers were dissolved in 1 ml of DMF solvent, and then 2 ml of distilled water was added to precipitate the copolymer NP. The average NP size ranges for the four types of copolymers (PLGA–PEG–OMe, PLGA–PEG–COOH, PLGA–PEG– Asp1 and PLGA–PEG–Asp3) were 122.9 ± 6.3, 166.3 ± 11.5, 144.1 ± 7.2 nm and 118.8 ± 7.5 nm, respectively, as indicated in Table 1. Clearly, the NP that were prepared in DMF were <200 nm in size. The polydispersity indices (PDI) of these particles were 0.094, 0.202, 0.136 and 0.149, respectively. These results demonstrate the narrow size distribution of the NP, as observed

Fig. 2. (a) 1H NMR spectrum and (b) ESI–MS spectrum of compound 6 (Asp3–(OMe)4) and (c) the FT-IR spectra of Asp3–(OMe)4, H2N–PEG–COOH and NH2–PEG–Asp3–(OMe)4.

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Fig. 3. 1H NMR spectrum of PLGA–PEG–(Asp)3.

in Table 1. In addition, the average zeta potentials of the PLGA– PEG–OMe NP and PLGA–PEG–ASP3 were 25.2 and 35.5 mV, respectively. However, the magnitude of the surface charge will depend on the acidic strengths of the surface groups and on the pH of the solution. The TEM images in Fig. 4 present a series of NP that were prepared using the nano-precipitation by solvent displacement method using PLGA–PEG–OMe, PLGA–PEG–COOH, PLGA–PEG– Asp1 and PLGA–PEG–Asp3, respectively. In this technique, the PLGA-based NP are stained by phosphotungstic acid, leaving the actual specimen untouched, and thus visible. The TEM samples revealed the presence of mostly spherical particles. Among them, the NP of PEG–PLGA conjugated with (Asp)n(1,3), appeared whole staining on spherical particles. In addition, the NP of PEG–PLGA without Asp moiety appeared ring staining on spherical particles. Each sample did not exhibit a broad size distribution; however, each observed particle size was in the previously determined particle-fraction range of 70–200 nm. To effectively deliver a drug to the targeted bone tissue, NP must have the ability to remain in the bloodstream for a considerable amount of time without being eliminated. NP are very rapidly opsonized in the bloodstream by phagocytic cells following IV administration. To avoid reticuloendothelial system (RES) uptake, the two most often-mentioned criteria are the formation of a hydrophilic surface using PEG and the use of an optimal particle size [33]. The NP used in a drug delivery system should be large enough to prevent their rapid leakage into blood capillaries (the pores in normal blood vessels are 2–6 nm in size), but small

enough to escape capture by fixed macrophages that are lodged in the RES; for example, the size of the sinusoid in the spleen and the fenestra of the Kuffer cells in the liver varies from 150 to 200 nm [34]. While larger NP are sequestered in the liver, consistent with observations in the spleen, very small NP (<70 nm) can pass through the sinusoidal fenestrations in the liver and be entrapped by underlying parenchymal cells [35]. Other factors should also be considered for drug-carrier NP. For example, their small size and large surface area can lead to particle aggregation, making the physical handling of NP difficult in liquid and dry forms. In addition, a small particle size and large surface area also readily result in limited drug loading and burst release. This work aimed to create bone targeting NP to transport a hydrophobic drug toolset that would enable the in vivo study of bone distribution. Consequently, the nanoparticles should be 70–200 nm in size to reach bone tissues by passing through the vascular structures and live organs of interest. 3.3. Bone mineral binding ability Apatite is the main component of bone in its natural state, i.e. the inorganic material of vertebrate bones, in which it is known as hydroxyapatite (Ca10(PO4)6(OH)2) or carbonated hydroxyapatite. A HAp binding assay was performed in vitro (Fig. 5a), and the affinity of the bone targeting NP for HAp is reported in Fig. 5b. Four types of NP (PLGA–PEG–OMe, PLGA–PEG–COOH, PLGA–PEG–Asp1 and PLGA–PEG–Asp3) were prepared by dialysis in sizes ranging from 100 to 200 nm in order to meet the size

Table 1 Average sizes, PDI and zeta-potential of the four types of nanoparticles precipitated by the solvent displacement method in various solvents. Copolymers

PLGA–PEG–OMe

PLGA–PEG–CM

PLGA–PEG–Asp1

PLGA–PEG–Asp3

Particle size (nm) PDI Zeta-potential (mV)

122.9 ± 6.3 0.094 25.2 ± 0.5

166.3 ± 11.5 0.202 –

144.1 ± 7.2 0.136 –

118.8 ± 7.8 0.149 35.5 ± 1.2

Note 1: Copolymers dissolved in 1 ml solvents, and then 2 ml distilled water was added into each solution to precipitate nanoparticles by solvent displacement method. Note 2: The PDI is an indication of variance in the sample: a low PDI (usually < 0.2) indicates that the sample is monodispersed.

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Fig. 4. TEM images of nanoparticles for (a) PLGA–PEG–OMe, (b) PLGA–PEG–COOH, (c) PLGA–PEG–Asp1 and (d) PLGA–PEG–Asp3.

requirement for long circulatory times. The data presented in Fig. 5b clearly demonstrate that the proportion of the PLGA– PEG–Asp3 NP bound to HAp (80%) was greater than the bound proportion of PLGA–PEG–Asp1 NP (45%). In particular, the affinity of the PLGA–PEG–(Asp)3 NP was more than three times that of the PLGA–PEG–OMe NP and PLGA–PEG–COOH NP. Wang et al. [14] confirmed that Asp (aspartic acid) prefers more crystalline HAp. However, these results indicate that the number and spatial distribution of the free carboxyl groups as well as the Aspn(1,3) structure allowed the NP to maintain their affinity for highly crystalline HAp. Moreover, it is not yet clear whether specific ligand–HAp interactions are involved, as these initial binding experiments do not distinguish between HAp affinity due to surface zeta potential and/or Asp3 specificity. Based on this evidence, the dendritic Asp3 moiety of the PLGA–PEG–Asp3 NP exhibited the best apatite mineral binding ability. It is also hypothesized that the bone affinity of a repeating sequence of acidic amino acids (Asp6) should be different from the present dendritic Asp3 moiety. 3.4. Cell viability and cell endocytosis The results of the MTS assay are presented as absorbance levels reflecting changes in the mitochondrial activity of the cells in response to a series of extracts from different materials. Fig. 6 presents the results of a direct cytotoxicity test for control groups administered blanks compared with groups administered 20 lg ml1 NP after incubation for 1 and 3 days. The control group did not exhibit cytotoxicity, and the absorbance of the

water-soluble formazan product value was highest on the fifth day. A similar phenomenon was observed with the other NP sample groups; thus, the four types of NP did not exhibit cell toxicity according to the MTS assay. In other words, the MTS assay revealed no significant differences between the control group and the sample groups. Furthermore, the water-soluble formazan absorbance values for all groups reflected cell viability for 5 days. It is generally accepted that in vitro cell–material interactions are a useful criterion in the evaluation of newly synthesized biomaterials. Bone-targeting NP are considered attractive carriers for drug delivery because of their minor toxic effects and their ability to associate with and be internalized into mammalian cells. Although NP do not travel into the cell via a specific pathway, the majority of NP have been observed to enter through macropinocytosis [36]. However, hybrid NP of PLGA–PEG–Asp3/FITC–PLGA–PEG–OMe (8/ 2) were observed in D1 BMSC after 10–20 min of incubation (Fig. 7). Hybrid NP were incubated with D1 cells; the FITC-labeled hybrid NP were internalized into the cells in a time-dependent manner and accumulated in a perinuclear pattern (co-staining of DAPI and FITC-labeled NP), indicating that the hybrid NP were internalized into the D1 cells. In addition, the hybrid NP were primarily endocytosed and did not localize to the plasma membrane. It is suggested that, in the D1 cells, the NP with an Asp3 moiety had a major effect on the endocytosis of the NP, the intracellular pathway, and the cells’ response to the NP. In addition, the conjugation with PEG–(Asp)3 did not affect the biocompatibility of PLGA. The animal experiments were used to assess the potential of bone-targeting in zebrafish and SD rats.

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Fig. 5. (a) HAp adsorption affinity experiments for the hydrophobic fluorescent dye of Prodan as a drug model and (b) their results.

Fig. 6. The cell viability of four types of nanoparticles and blank were evaluated by MTS assay.

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Fig. 7. (i–iii) Time-dependent fluorescence imaging of PLGA–PEG–Asp3/FITC–PLGA–PEG–OMe (8/2) NP, (iv–vi) DAPI staining and (vii–ix) merge images, which transport into D1 living cells.

Fig. 8. Zebrafish larvae of 7 dpf were injected with nanoparticles colloids containing synthetic FITC-labeled hybrid nanoparticles (green fluorescence) and TexasRed (red fluorescence) at the common cardinal vein (white arrow head) and observed at (a) 0.5 hpi and (b) 24 hpi. Displayed are the dorsal views of larvae imaged under a fluorescence dissecting microscope with anterior to the top. op, opercle; cl, cleithrum. ASP3 consists of hybrid nanoparticles of PLGA–PEG–Asp3/FITC–PLGA–PEG–OMe (8/2), and OMe consists of hybrid nanoparticles of PLGA–PEG–OMe/FITC–PLGA–PEG–OMe (8/2).

3.5. Bone-targeting nanoparticles in vivo 3.5.1. Bone-targeting nanoparticles in zebrafish in vivo To prevent NP with sizes >200 nm from causing vascular obstruction, the as-received hybrid NP solution was filtered using

a 0.2-lm membrane filter before injecting the solution into zebrafish. The hybrid NP of PLGA–PEG–Asp3/FITC–PLGA–PEG–OMe (8/2) and PLGA–PEG–OMe/FITC–PLGA–PEG–OMe (8/2) accumulated in the bone of the zebrafish larvae 24 hpi. No significant difference in the pattern of hybrid NP deposition was observed between the

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Fig. 9. Optical photographs and fluorescence images of (II) IR780/PLGA–PEG–Asp3 nanoparticles and (I) IR780/PLGA–PEG–OMe nanoparticles taken 48 h after nanoparticle transport into SD rats. (a) Optical photographs and (b) fluorescence images of femur/tibia bone; (c) fluorescence images of vertebrae bone, and (d) fluorescence images of cross-section of vertebrae bone.

PLGA–PEG–OMe NP group and the PLGA–PEG–Asp3 NP group injected at 0.5 hpi (Fig. 8a, green fluorescence). Both groups contained evenly distributed green fluorescent signals (corresponding to hybrid NP), and a comparable pattern of red fluorescence (reflecting the injected TexasRed) was observed in both the experiment and control groups, indicating the successful and equivalent injection of the hybrid NP solutions into the larval circulation of both groups. No appreciable or specific accumulation of the green fluorescent signal was detected in either the PLGA–PEG–OMe or PLGA–PEG–Asp3 groups at 0.5 hpi. Conversely, green fluorescence was observed at 24 hpi in the larval cleithrum and opercle only in the PLGA–PEG–Asp3 NP injected larvae (Fig. 8b). These data demonstrate that the PLGA–PEG–Asp3 NP can specifically target and accumulate in the mineralized bone of zebrafish larvae. 3.5.2. Bone-targeting nanoparticles in rats in vivo An in vivo biodistribution assay in SD rats was performed to further examine the bone targeting ability of the PLGA–PEG–Asp3 NP. Fig. 9 shows the accumulation of the NP in the skeleton (represented by the bilateral femur/tibia and vertebrae). However, the as-received NP colloids were not passed through a 0.2-lm membrane filter before injection into the rats. The fluorescence intensities were evaluated because both NP groups (IR-780/ PLGA–PEG–OMe NP and IR-780/PLGA–PEG–Asp3 NP) reached the skeleton 48 h after vein administration via the rat tail. It is clear that the fluorescence intensities of IR-780 in the bone due to the

PLGA–PEG–Asp3 NP were higher than those due to the PLGA– PEG–OMe NP control after 48 h. However, more in vivo tests, such as bone targeting and whole body biodistribution, are needed to truly determine the efficacy of these targeted nano-polymeric delivery systems. The fluorescence signals were present mainly in the lungs of the rats, except for liver, bilateral femur/tibia and vertebrae from the PLGA–PEG– OMe NP control groups. However, the fluorescence signals were barely detectable in the spleen, kidneys and heart of the rats from the PLGA–PEG–OMe NP control groups (Fig. S5a, c in the supporting information). However, the fluorescence signals were observed mainly in the bilateral femur/tibia, vertebrae and liver of the rats from the PLGA–PEG–Asp3 NP treatment groups. The fluorescence signals were also barely detectable in the hearts, lung, kidneys and spleens of the rats from of the PLGA–PEG–Asp3 NP treatment groups. (Fig. S5a, b in the supporting information). Furthermore, the data were quantified from the fluorescence intensities using IVIS 200 software, and the results in Fig. 10 are also consistent with the biophotonic imaging results. The tissue distributions of the fluorescence intensities clearly indicate that 43.9% more IR-780/ PLGA–PEG–Asp3 NP were present in the bone area (bilateral femur/tibia and vertebrae) than in the case of the IR-780/PLGA– PEG–OMe NP. Therefore, the dendritic trimer in the aspartic acid oligopeptide (Asp3) conjugated PLGA–PEG copolymer has a tendency to target and accumulate in bone. The fluorescence intensities in the liver and lung tissue also remained, indicating that the

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Fig. 10. Tissue distribution graphically represented as a percentage of fluorescence intensity detected 48 h after nanoparticle transport into SD rats. The data are plotted as means ± SD (n = 3), ⁄ indicates p < 0.05. (Note: bone including bilateral femur/tibia and vertebrae.)

NP also accumulated in the liver and lungs. The NP may have appeared in the liver, because heparin is metabolized by heparinase, which is located in the liver, and IR-780 is metabolized through the liver [37]. Moreover, the Kupffer cells in the liver play a role in uptake and extra-phagocytosis degradation [38]. It is worth mentioning that the majority of PLGA–PEG–OMe NP are initially trapped inside the lungs after IV infusion for 48 h; the average fluorescence signal from the lungs was significantly higher than lungs of PLGA–PEG–ASP3 NP. Results showed that PLGA– PEG–ASP3 NP can reduce NP trapping in lungs and increased the NP passage through the lung capillaries of rat, despite the relatively small differences in size being obtained between PLGA–PEG–ASP3 NP (118.8 ± 7.5 nm) and PLGA–PEG–OMe NP (122.9 ± 6.3 nm). But, the zeta potential data for PLGA–PEG–OMe NP (25.2 mV) were also more weakly negative than PLGA–PEG– ASP3 NP (35.5 mV). Therefore, the electrical interaction between the weaker negative zeta potentials of PLGA–PEG–OMe NP might have potentiated the accumulation of PLGA–PEG–OMe NP aggregates in the lung capillaries. The underlying mechanism(s) of the lung accumulation of NP were not pursued in detail in the present study, but the transient formation of large NP aggregates in contact with plasma proteins during circulation, followed by enhanced entrapment of large aggregates by the lung capillaries, might represent the principal mechanisms. This hypothesis is based on the fact that large size aggregates (e.g. 2.0–10.0 lm) are efficiently trapped in the capillary bed of the normal vasculature of the lung [39]. From the in vivo preliminary data, the two animal model biodistribution studies in zebrafish and rats showed significant differences compared with the non-targeted (without Asp3 oligopeptides) controls; the bone-targeting oligopeptides containing dendritic Asp3 can be expected to reduce the drug administration dose, achieve higher bone tissue drug accumulation and reduce side effects in other organs caused by chemotherapy drugs. Meanwhile, any bone diseases in the rat model may require further analysis to determine the toxicity of this system, including therapeutic drugs, in the future.

bone targeting ability. It was confirmed that the moieties and final products of Asp1–(OMe)2, Asp3–(OMe)4, PEG–(Asp)1,3–OMe, PLGA– PEG–OMe, PLGA–PEG–COOH and PLGA–PEG–(Asp)1,3 were synthesized successfully using 1H NMR or ESI–MS. However, PEG–Asp3 was identified by its FT-IR spectrum, because its 1H NMR signals were unclear. It was confirmed that the average particle size was <200 nm for the four types of copolymer NP and that the dendritic Asp3 moiety of the PLGA–PEG–(Asp)3 NP had enhanced bone-binding capacity when binding to apatite mineral surfaces, because of its affinity for HAp. In addition, the PLGA–PEG–Asp3 NP were taken up by D1 bone marrow stem cells without causing significant cell toxicity. Biophotonic imaging technology was also used to examine the organ distribution of the FITC-labeled areas or of the IR-780 fluorescent dye delivered by the NP with or without the Asp3 moiety in zebrafish and SD rats. It was observed that the intensity of the intraosseous fluorescence signal was stronger after injection with the PLGA–PEG–Asp3 NP than after treatment with the PLGA–PEG–OMe NP. The bone-targeting NP based on the dendritic trimer in the aspartic acid oligopeptide (Asp3) conjugated to PLGA– PEG copolymers have the ability to target bone and accumulate there; thus, their use for the treatment of bone diseases in the future is proposed. In particular, OP, anticancer and antibacterial agents could take advantage of this therapeutic strategy. Acknowledgments The authors gratefully acknowledge the support for this research by the Ministry of Science and Technology in Taiwan (NSC-101-2628-E-037-002), and Ministry of Economic Affairs in Taiwan (101-EC-17-A-19-S1-176). Appendix A. Figures with essential color discrimination Certain figures in this article, particularly Figs. 1–3, 5–9 are difficult to interpret in black and white. The full color images can be found in the on-line version, at http://dx.doi.org/10.1016/ j.actbio.2014.07.015.

4. Conclusions

Appendix B. Supplementary data

The present authors designed, synthesized and compared four types of amphoteric copolymers (PLGA–PLGA–OMe, PLGA–PEG– COOH, PLGA–PEG–(Asp)n(1,3)) and then prepared NP to assess their

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2014. 07.015.

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