Osteoblast-Targeting-Peptide Modified Nanoparticle for siRNA

May 13, 2016 - Department of Oral Implantology, School of Stomatology, Tongji University, Shanghai 200072, China. § The First Affiliated Hospital of ...
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Osteoblast-Targeting-Peptide Modified Nanoparticle for siRNA/microRNA Delivery Yao Sun,‡,∥,⊥,# Xiongzhen Ye,†,# Mingxiang Cai,‡,# Xiangning Liu,§,# Jia Xiao,† Chenyang Zhang,‡ Yayu Wang,† Li Yang,† Jiafan Liu,† Shannai Li,† Chen Kang,⊥ Bin Zhang,⊥ Qi Zhang,‡ Zuolin Wang,*,‡,∥ An Hong,*,† and Xiaogang Wang*,† †

Department of Cell Biology & Institute of Biomedicine, College of Life Science and Technology, Jinan University, Guangzhou 510632, China ‡ Department of Oral Implantology, School of Stomatology, Tongji University, Shanghai 200072, China § The First Affiliated Hospital of Jinan University, Guangzhou 510632, China ∥ Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai 200072, China ⊥ Sino-Russian Institute of Hard Tissue Development and Regeneration, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086, China S Supporting Information *

ABSTRACT: Antiosteoporosis gene-based drug development strategies are presently focused on targeting osteoblasts to either suppress bone loss or increase bone mass. Although siRNA/microRNA-based gene therapy has enormous potential, it is severely limited by the lack of specific celltargeting delivery systems. We report an osteoblast-targeting peptide (SDSSD) that selectively binds to osteoblasts via periostin. We developed SDSSD-modified polyurethane (PU) nanomicelles encapsulating siRNA/ microRNA that delivers drugs to osteoblasts; the data showed that SDSSD−PU could selectively target not only bone-formation surfaces but also osteoblasts without overt toxicity or eliciting an immune response in vivo. We used the SDSSD−PU delivery system to deliver anti-miR-214 to osteoblasts and our results showed increased bone formation, improved bone microarchitecture, and increased bone mass in an ovariectomized osteoporosis mouse model. SDSSD−PU may be a useful osteoblasttargeting small nucleic acid delivery system that could be used as an anabolic strategy to treat osteoblast-induced bone diseases. KEYWORDS: osteoblast targeting peptide, polyurethane nanoparticle, gene delivery, osteoporosis therapy, bone formation

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However, clinical success has been limited due to nucleic acid biodegradation and lack of target tissue- or cell-specificity of the delivery system. Another key challenge of gene therapy is the development of safe and effective delivery systems. Both viral and nonviral vectors have been used to deliver nucleic acids;8 but there are substantial challenges limiting viral vectors use, including safety and immunogenicity issues.9,10 In contrast, nonviral vectors have lower apparent immunogenicity and diverse nonviral vectors have been used to deliver therapeutic nucleic acids to the bone microenvironment. However, these vectors have off-target effects, and are often not biodegradable or biocompatible, or have low-encapsulation efficiency.

keletal mass is regulated by hematopoietic derived osteoclast resorption and mesenchymal derived osteoblast formation.1 Imbalance in osteoblast and osteoclast differentiation and function results in skeletal diseases including osteoporosis, rheumatoid arthritis, and bone cancer metastases.1 Osteoporosis is a systemic bone metabolism disorder that affects individuals worldwide, increasing bone fragility and fracture risks.2 However, clinical anabolic drugs that enhance bone formation are limited to full-length parathyroid hormone (PTH 1-84) or its N-terminal fragment, teriparatide (PTH 134);3,4 however, both drugs have severe side effects such as nausea, diarrhea, and hypercalcemia.5,6 Thus, an osteoblast- or osteoclast-targeted drug delivery system is necessary for treating osteogenic disorders. Gene therapy is an innovative strategy for modulating gene expression to treat disease by delivering exogenous small nucleic acids such as DNA, RNA, short interfering RNA (siRNA), or microRNA (miRNA).7 © 2016 American Chemical Society

Received: December 12, 2015 Accepted: May 13, 2016 Published: May 13, 2016 5759

DOI: 10.1021/acsnano.5b07828 ACS Nano 2016, 10, 5759−5768

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Figure 1. Selection and identification of an osteoblast targeting peptide. (A) Scheme for the selection of mouse and human osteoblasts specific binding peptides by phage display technology. (B) Repetitive SDSSD regions in the amino acid sequence of mouse dentin phosphoprotein. (C) Targeted binding of FITC-SDSSD peptides to mouse and human osteoblasts observed by microscopy. Scale bar, 10 μM. (D) Targeted binding of SDSSD peptides to mouse and human osteoblasts detected by flow cytometry. (E) Fluorescence micrographs of mouse and human osteoblasts, bone marrow mesenchymal stem cells (BMSCs), osteoclasts, and bone marrow cells after incubation with FITC-SDSSD peptides for 0.5 h. Scale bar, 10 μM. (F) Flow cytometry analysis of the binding ability of the SDSSD peptide to mouse and human osteoblasts (OBs), bone marrow mesenchymal stem cells (BMSCs), osteoclasts (OCs), and bone marrow cells (BMCs).

Here, we report an osteoblast-targeted delivery system for osteoporosis gene therapy. Phage display was used to select peptides that could target both mouse and human osteoblasts, and we identified the selected peptide sequence as Ser-Asp-SerSer-Asp (SDSSD). Using affinity chromatography and cellbinding experiments, we showed that SDSSD peptide had binding affinity for periostin (also known as osteoblast-specific factor 2, OSF-2), indicating that SDSSD peptide targeted osteoblasts in a ligand−receptor specific manner. Thus, we postulated that SDSSD peptide could selectively target osteoblasts. Next, we designed and synthesized PU nanomicelles modified with SDSSD peptide as our osteoblasttargeted delivery system and performed in vitro and in vivo studies to confirm that targeted delivery of nucleic acids to osteoblasts was successful.

Consequently, improved and more efficient osteoblast-targeted nonviral delivery systems are required. Nanoparticle modification of the targeting ligand can improve distribution11 by modifying key attributes such as specificity and stability. Peptides are attractive targeting ligands due to potentially low immunogenicity, high avidity, easy bioconjugation and synthesis, and lower costs.12 Small molecule-targeting moieties such as endogenous folate, sugar, and carbohydrates have high receptor binding affinity, but their receptors are widely expressed; thus, they are nonselective and nonspecific in targeting tissues.13,14 Exogenous ligands such as proteins and monoclonal antibodies are potentially immunogenic and are rapidly cleared, and have engineering challenges for scale-up and manufacturing.15 Additionally, aptamer stability may be affected by heat, exonuclease or endonuclease degradation, and other environmental factors,16 so their efficacy and applicability are limited.17 Moreover, nanoparticles of 20− 200 nm offer prolonged nucleic acid encapsulation circulation as they are too large for renal filtration and evade phagocytic clearance, reducing nontargeted drug distribution.11,18 Innovative designs such as nontraditional materials to reduce cytotoxicity or conjugation with functional ligands may offer advantages over conventional nanoparticle delivery systems.19 Unlike traditional lipid-based nanoparticles, polyurethane (PU) nanomicelles encapsulate small nucleic acids via electrostatic interactions, and previous clinical results have confirmed excellent biocompatibility, low cytotoxicity, and good mechanical flexibility.

RESULTS Selection and Identification of an Osteoblast Targeting Peptide. To identify an osteoblast-targeting peptide, phage technology was used to screen peptides with high binding affinity for both mouse and human osteoblasts (Figure 1A). A five-amino acid motif, Ser-Asp-Ser-Ser-Asp (SDSSD) was present in multiple copies in the candidate peptides. Interestingly, this SDSSD sequence was highly repeated in dentin phosphoprotein protein (DPP) amino acid sequence, which is a biomineralization related protein highly expressed in tooth and also in bone (Figure 1B). To determine the osteoblast-targeting ability of the SDSSD peptide, mouse and 5760

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ACS Nano human osteoblasts were used in cell binding experiments, and the SDSSD peptide bound well to osteoblasts; in comparison, DSS6, which was previously reported to have good affinity for the bone-formation surface, had weaker fluorescence intensity and greater cytotoxicity (Figure 1C, Figure S1A,B). Flow cytometry analysis also showed that SDSSD peptide targeted osteoblasts because it had the highest fluorescence intensity compared with other control groups (Figure 1D). Bone-derived cells such as BMSCs, osteoclasts, and bone marrow cells were used to confirm the binding specificity of the SDSSD peptide for osteoblasts. We observed that osteoblasts had the highest fluorescence signals compared with other cell types (Figure 1E). Flow cytometry data confirmed that the SDSSD peptide bound with high affinity to both mouse and human osteoblasts (Figure 1F) and that this peptide was not cytotoxic in vitro (Figure S1C,D). SDSSD Peptide Binding to Periostin for OsteoblastTargeting. To explore the osteoblast-targeting mechanism of the SDSSD peptide, proteins captured by SDSSD peptide were collected and identified. With the use of mass spectrometry and pulldown assay, a single protein with the molecular weight near 90 kDa was identified as periostin which is also called osteoblast-specific factor 2 (OSF-2) (Figure 2A, Supporting Information Tables S1 and S2) and confirmed by Western blot in both human and mouse osteoblasts (Figure 2B). To investigate whether SDSSD peptide targeting osteoblasts was driven by periostin binding, FITC-SDSSD was incubated with mouse and human osteoblasts and we observed numerous colocalizations of SDSSD peptide with periostin in both osteoblast species as demonstrated by confocal microscopy (Figure 2C). In addition, competition assays verified that periostin had a role in SDSSD targeting to osteoblasts. Osteoblasts pretreated with SDSSD peptide to block periostin had lower FITC-SDSSD-periostin binding activity than osteoblasts incubated with FITC-SDSSD directly and without pretreatment (Figure 2D). Design, Synthesis, and Characterization of SDSSD− PU. Thus far, our data indicated that the SDSSD peptide could target osteoblasts safely and specifically via a periostin-mediated mechanism; therefore, we used the SDSSD peptide to modify nanoparticles for the development of an osteoblast-targeting small nucleic acid delivery system. Polyurethane (PU) was chosen as a vector as it is a biomedical material with ease of use for SDSSD peptide conjugation. Briefly, a series of monomers were synthesized and PU was prepared via a three-step polymerization reaction (Figure S2A). Prepared PU selfassembled into nanomicelles which were then conjugated to the SDSSD peptide to form SDSSD−polyurethane (SDSSD− PU). Infrared spectroscopy (IR) data confirmed successful conjugation of the SDSSD peptide to PU (Figure S2B). The successful formation of SDSSD peptide to conjugate with PU was also confirmed by UV−vis spectroscopic characterization (Figure S2C). The amount of peptide on PU was determined to be 6% SDSSD found on conjugate to PU (Figure S2D,E).With transmission electron microscopy (TEM), we observed that most nanoparticles were uniform in size and morphology (Figure 3B). The nanoparticle size and zeta potential of SDSSD−PU were assessed using laser dynamic light scattering (DLS) and it was determined that the average diameter of SDSSD−PU is 70 nm (Figure 3C) and the average zeta potential is 16 mV (Figure 3D). The nucleic acid binding capacity of SDSSD−PU nanomicelles was assessed by agarose gel electrophoresis, and the data indicated that nucleic acid

Figure 2. SDSSD peptide binding to periostin for osteoblast targeting. (A) The identified top five proteins binding to the SDSSD peptide identified by mass spectrometry. (B) A representative Western blot of the selected proteins binding to SDSSD peptide after affinity purification. (C) Co-localization of the SDSSD peptides and periostin in mouse and human osteoblasts in vitro. The nuclei were counterstained with DAPI. Scale bar, 10 μM. (D) Competition assays. (a) Mouse osteoblasts were incubated with FITC-SDSSD directly for 6 h. (b) After 1 h of preincubation with SDSSD, mouse osteoblasts were further incubated with FITCSDSSD for further 6 h. (c) Human osteoblasts were incubated with FITC-SDSSD directly for 6 h. (d) After 1 h of preincubation with SDSSD, human osteoblasts were further incubated with FITCSDSSD for 6 h. Scale bar, 10 μM.

mobility was retarded by SDSSD−PU nanomicelles at weight ratios of SDSSD−PU/nucleic acid (N/P, w/w) greater than 10:1 (Figure 3E). To test the resistance of entrapped nucleic acid to serum-mediated degradation, free nucleic acid and SDSSD−PU-nucleic acid complexes were incubated with fetal calf serum. Electrophoretic assays indicated that a band representing free nucleic acid was absent after incubation with serum for 6 h, but the SDSSD−PU-nucleic acid band was still visible after incubation with serum for 24 h (Figure 3F). Targeted Delivery of siRNA to Osteoblasts in Vitro. To investigate whether prepared SDSSD−PU could specifically deliver siRNA to osteoblasts in vitro, mouse and human cells from the bone environment were sorted by flow cytometry and were subsequently incubated with FAM-labeled siRNA delivered by SDSSD−PU. Strong fluorescence signals were observed in osteoblasts compared with other cell types (Figure 5761

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necrosis factor-α (TNF-α), and interferon-α (IFN-α) (Figure S3F). Number of mice is six in each group. To investigate whether SDSSD−PU could deliver siRNA to bone-formation surfaces, we assessed localization of siRNA delivered by PU or SDSSD−PU. Bone-formation surfaces (labeled with xylenol orange, a red fluorescent calcium-binding dye that labels new bone deposition at bone-formation surfaces, and pointed by arrows) were abundantly colabeled with siRNA delivered by SDSSD−PU, whereas few instances of such colabeling were observed when siRNA was delivered by PU (Figure 4D). We next determined whether SDSSD−PU could deliver siRNA to osteoblasts in vivo by analyzing colocalization of FAM-siRNA with DMP1 (a mature osteoblast marker, pointed by arrows). Numerous instances of colocalization of FAM-siRNA with DMP1-positive cells of mice treated with SDSSD−PU were observed, whereas few instances of such overlapping staining were observed in the PU groups (Figure 4E). In addition, consistent with in vitro results (Figure 2B), the colocalization of FAM-siRNA with periostin in osteoblasts further confirmed the targeting of SDSSD through periostin of cell (Figure 4F). GFP-siRNA Knockdown Efficiency Delivered by SDSSD−PU in GFP Transgenic Mice. To evaluate gene knockdown efficiency of SDSSD−PU in vivo, GFP transgenic mice were administered SDSSD−PU−GFP siRNA (via tail vein injection), and GFP fluorescence signals in organs were measured by biophotonic imaging technology. Weaker fluorescent signals were observed in bone after treatment with SDSSD−PU−GFP siRNA (Figure 5A). GFP mRNA expression was measured in tissues with real-time PCR. We found that the decrease of GFP mRNA levels in bone was more significant than in other nonskeletal tissues (Figure 5B). Western blot analysis confirmed that GFP protein decreased markedly after treatment with SDSSD−PU−GFP-siRNA (Figure 5C) and minimal levels of GFP fluorescence were detected in osteoblasts of animals treated with SDSSD−PUGFP-siRNA (Figure 5D). Then, we measured GFP-siRNA knockdown efficiency in osteoblasts sorted by flow cytometry from mice treated with different siRNAs. Compared with control siRNA, the knockdown efficiency of GFP-siRNA was significantly higher in osteoblasts, as reflected by reduced GFP mRNA levels (Figure 5E). Similarly, Western blot analysis confirmed that SDSSD−PU−GFP-siRNA had greater protein knockdown efficiency (Figure 5F). Antiosteoporotic Efficacy of SDSSD−PU-Anti-miR214. Anti-miR-214 is reported to increase osteoblast activity in vivo. Thus, we studied the therapeutic effects of anti-miR-214 in an ovariectomized mouse model of osteoporosis (OVX). First, we noted that in osteoblasts miR-214 decreased 80% after SDSSD−PU-anti-miR-214 treatment (Figure 6A). In assessing bone formation with calcein labeling, we noted that compared with mice treated with SDSSD−PU-anti-scramble, a wide band was observed in mice treated with SDSSD−PU-anti-miR-214 (Figure 6B). In addition, bone microarchitecture and bone mineral density (BMD) were measured with computed tomography (micro-CT). Improved bone microarchitecture and greater bone mass were found in mice treated with SDSSD−PU-anti-miR-214 (Figure 6C). Micro-CT quantification and histomorphometric analysis confirmed that OVX + SDSSD−PU-anti-miR-214-treated animals had significantly increased BMD and mineral apposition rate compared with the OVX + SDSSD−PU-anti-scramble group after treatment (Figure 6B,D).

Figure 3. Design, synthesis and characterization of SDSSD−PU. (A) Schematic diagram of SDSSD−PU. (B) Typical image of transmission electron micrograph of SDSSD−PU. Scale bar, 100 nm. (C) The particle size distribution of SDSSD−PU with an average size of 70 nm. (D) Zeta potential of SDSSD−PU determined by dynamic light scattering. (E) The binding capacity of SDSSD−PU to siRNA measured by electrophoretic mobility assays. (F) Serum stability of free siRNA and SDSSD−PU-siRNA complexes analyzed by gel electrophoresis after incubation with serum at the indicated time points.

4A). Additionally, cell viability was unaffected following incubation with SDSSD−PU (Figure S3A,B). We then studied whether specific targeting of SDSSD−PU was driven by SDSSD peptide in osteoblasts. Compared with cells incubated with PU, a mixture of SDSSD and PU or preincubated with SDSSD peptide to block periostin, cells incubated with SDSSD−PU had the most internalization of siRNA in mouse and human osteoblasts (Figure 4B). Specific Localization of siRNA Delivered by SDSSD− PU in Vivo. Hemagglutination assays confirmed that no agglutination occurred when erythrocytes were incubated with PU or SDSSD−PU, indicating that both were compatible (Figure S3C). To identify the location of siRNA delivered by SDSSD−PU in vivo, mice were intravenously (iv) administered fluorescently labeled siRNA delivered by SDSSD−PU or PU, and biophotonic imaging techniques were used to examine tissue distribution of FAM-labeled siRNA. Fluorescence intensity in bone tissues of the SDSSD−PU group was greater than the PU group (Figure 4C). However, fluorescence intensity of siRNA in liver, kidney and, other organs was lower after treatment with SDSSD−PU-siRNA (Figure 4C). Toxicity assays in vivo demonstrated that both SDSSD−PU and PU did not have off-target effects (hearts, liver, spleen, and kidney Figure S3D). There were no statistically significant differences in creatine kinase-MB (CK-MB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) (Figure S3E). Moreover, administration of SDSSD−PU or PU did not elicit an inflammatory response, as reflected by unaltered levels of inflammatory cytokines including interleukin-6 (IL-6), tumor 5762

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Figure 4. Specific localization of siRNA delivered by SDSSD−PU in vitro and in vivo. (A) Specific uptake of SDSSD−PU-siRNA by mouse and human cells from the bone environment examined by microscopy. Scale bar, 20 μM. (B) SDSSD peptide-dependent siRNA delivery to mouse and human osteoblasts by SDSSD−PU. Scale bar, 20 μM. (C) Tissue-selective distribution of siRNA delivered by SDSSD−PU in mouse organs and bone. The concentration of SDSSD−PU is 1 mg/mL, and 0.2 mL/(mice/injection). (D) Localization of siRNA to mouse bone formation surfaces delivered by SDSSD−PU or PU. Scale bar, 20 μM. (E) Localization of siRNA in mouse local osteoblasts delivered by SDSSD−PU. Scale bar, 20 μM. (F) Localization of Periostin in mouse local osteoblasts. Scale bar, 20 μM.

DISCUSSION To generate a delivery system that can directly target osteoblasts for osteogenic gene delivery, numerous molecules have been used to target bone,20,21 such as tetracycline,22 bisphosphonate,23 oligopeptide,24−26 and aptamer.27 Evidence suggests that tetracycline binds strongly to bone apatite surfaces likely due to chelation of tetracycline with calcium ions on the hydroxyapatite surface.28−30 However, the complicated chemical structure and the poor stability during chemical modification of tetracycline may limit its use as a bonetargeting moiety.21 Bisphosphonates also bind strongly to the hydroxyapatite surface, and the binding affinity of bisphosphonates for hydroxyapatite was mediated by a P−C−P bond.31 Although bisphosphonates have the potential for bonetargeting therapeutic use, the drugs are distributed to both bone-formation and bone-resorption surfaces, leading to less specificity. Bisphosphonates also have side effects such as bone turnover suppression and increased risk for atypical femur fractures.32,33 Compared with tetracycline and bisphosphonates, (DSS)n peptides target hydroxyapatite by favorably binding to less crystallized hydroxyapatite. DSS6, a representative targeting oligopeptide, has been reported to preferentially bind to boneformation surfaces.26,34 The CH6 aptamer, a single-stranded oligonucleotide modified with 2′-O-methyl-nucleotide, can directly target osteoblasts at the cellular level.27 However, applications for typical aptamers are limited by nuclease degradation and high costs.16,35 In our study, SDSSD peptide could directly target both mouse and human osteoblasts and offered specific and efficient binding (Figures 1 and 2). Additionally, SDSSD was less toxic than DSS6 peptide (Figure S1), which suggests that SDSSD likely has fewer side effects.

Mechanistically, the SDSSD peptide was specifically targeted to osteoblasts via a periostin-mediated mechanism, as evidenced by colocalization of SDSSD peptide with the periostin protein (Figure 2C and Figure 4F), and by the fact that blocking of periostin by preincubation with SDSSD peptide significantly decreased the SDSSD peptide internalization (Figure 2). Similar data were obtained with a SDSSD− PU complex: periostin preincubated with SDSSD peptide was not available to interact with the SDSSD−PU complex, resulting in less internalization of the complex, which suggests that the delivery efficiency of SDSSD−PU is regulated by periostin on osteoblast membrane (Figure 4). The SDSSD peptide specificity for osteoblasts is driven by preferential expression of periostin by osteoblasts.36 In 30 min incubation test, the osteoblasts could bind much more SDSSD than BMSCs or other related cells (Figure 1E and Figure 4A). If the observation time was extended, the interaction of SDSSD to BMSCs will increased slightly. Also, a human endothelial cells line (HUVEC) is used as a control to ensure that the particles are not internalized unspecifically by blood vessel endothelial cells when injected systemically. Endothelial cells also take much less miRNAs compared to osteoblasts (Figure S4). In addition, DSPP is a mineralized tissue matrix protein which is expressed in dentin and bone. Dentin phosphoprotein (DPP) is a processed COOH-fragment from dentin sialophosphoprotein (DSPP). It is a highly phosphorylated protein, which could promote dentin formation and mineralization under specific pathway. The highly repeated SDSSD sequence is a unique character of DPP amino acid sequence. More attention should be paid for the specifically binding between SDSSD and cells. The reaction mechanism of this short peptide 5763

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As a consequence of specific SDSSD/periostin interactions, SDSSD−PU specifically targeted osteoblasts had high gene knockdown efficiency and enhanced bone anabolic activity. Immunohistochemical data indicated that SDSSD−PU targeted bone-formation surfaces and osteoblasts at the cellular level, as evidenced by colocalization of nucleic acids delivered by SDSSD−PU with bone formation surface and osteoblast markers (Figure 4). In addition, osteoblast-selective knockdown of the GFP gene corresponded to bone tissue and osteoblast-selective distribution of nucleic acids delivered by SDSSD−PU (Figure 5). Furthermore, micro-CT data from OVX mice indicated that SDSSD−PU-anti-miR-214 significantly improved bone formation and increased bone mass (Figure 6). The stability of nanoparticles was also tested in vivo and in vitro, and during the whole observation period, the results showed that the stability meets the requirements of drug delivery system (Figure S5A−C) and the degradation mechanisms of PU mainly lysosomal degradation pathway (Figure S5D).

CONCLUSIONS In this study, we have reported the design, synthesis, and evaluation of the SDSSD−PU micelle nanoparticle for delivering siRNA/microRNA to osteoblasts and exerting a therapeutic benefit by RNAi activity. We demonstrated that SDSSD−PU could target osteoblasts both in vitro and in vivo as an osteoblast-targeting delivery system safely and effectively. The results of this study indicated a tremendous potential of SDSSD−PU nanoparticles for treating osteoporosis and osteoblast dysfunction-induced metabolic syndromes.

Figure 5. GFP-siRNA knockdown efficiency in GFP transgenic mice treated with SDSSD−PU. (A) Representative fluorescence images of organs from GFP transgenic mice treated with SDSSD− PU−GFP-siRNA. (B) GFP mRNA level in mouse organs determined by real-time PCR. Data shown as the mean ± SD. N = 6 per group. (C) GFP protein levels in mouse organs determined by Western blot. (D) Fluorescence images of mouse bone formation surface treated with SDSSD−PU−GFP-siRNA. Scale bar, 20 μM. (E) GFP mRNA level in isolated osteoblasts from mice treated with SDSSD−PU−GFP-siRNA. Data shown as mean ± SD.N = 6 per group. (F) GFP protein levels in isolated osteoblasts from mice treated with SDSSD−PU−GFP-siRNA determined by Western blot. OB = osteoblast; BM = bone marrow.

METHODS Cell Culture. Mouse osteoblast cell line MC3T3-E1 clone 4 was cultured in alpha-modified Eagle’s medium (α-MEM, Life Technologies) containing 10% fetal bovine serum (FBS) (Life Technologies) and 1% penicillin and streptomycin (Invitrogen). Human osteoblast cell line hFOB 1.19 was cultured in DMEM (Invitrogen) with 10% FBS and 1% penicillin and streptomycin. All cells were maintained in 5% CO2 at 37 °C. Phage Display. Phage display was performed as described previously.37 Briefly, a Ph.D.-12 Phage Display Peptide Library (Ph.D.-12 Phage Display Peptide Library Kit, New England Biolabs, Beverly, MA) was incubated with target cells for 2 h at 37 °C with gentle agitation. After a washing step, the bound phages were eluted and then incubated with nontarget cells for 2 h at 37 °C. The supernatant containing phages was obtained and amplified in Escherichia coli. After multiple rounds of biopanning, the enriched pool of phages that bound to target cells without binding to nontarget cells were sequenced by PCR, and the DNA sequence was translated to the displayed peptide. Peptide Binding Assays. The peptide binding assay was performed according to previous methods.38 Peptide conjugated FITC was incubated with cells at a final concentration of 20 μg/mL for 0.5 h at 37 °C. Then, unbound peptides were removed from the well by washing three times with PBS and the nuclei were stained with DAPI. Finally, the binding ability of peptides was analyzed using microscopy and flow cytometry. Screening and Identification of the Potential Receptor of the Selected Peptide. Pull-down experiments were used to screen the potential receptor of the SD peptide, and mass spectrometry and Western blot analysis were used to identify the receptor.39,40 SD peptide was incubated with EZ Link Sulfo-NHS-SS-Biotin overnight, and the reaction mixture was purified by dialysis to remove excess biotin. Osteoblasts pellets were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, and 25 mM Tris-HCl, pH 7.4), and insoluble proteins, DNA

Figure 6. Antiosteoporosis efficiency of SDSSD−PU-anti-miR-214. (A) Real-time PCR analysis of miR-214 levels in isolated osteoblasts collected from the groups of mice indicated. Data shown as the mean ± SD. N = 6 per group. (B) Representative images showing new bone formation by double calcein labeling in each group. Scale bar, 20 μM. (C) Representative images showing three-dimensional trabecular architecture by micro-CT reconstruction in the distal femurs. Scale bar, 1 mm. (D) Micro-CT measurements for BMD and histomorphometric analysis for MAR in the distal femurs. Data shown as the mean ± SD. N = 6 per group.

found in this study is also a key issue to further illustrate the function of this extracellular matrix protein. 5764

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ACS Nano and cell debris were cleared by centrifugation at 15 000g at 4 °C for 20 min. The biotin−peptides were incubated with osteoblast lysates overnight at 4 °C, and then the mixture was incubated with NeutrAvidin beads for 4 h at 4 °C. Beads were washed four times with PBS containing 5% acetonitrile, and the captured biotinylated peptides complexes were eluted with eluent solution (0.2% TFA, 0.1% formic acid, and 80% acetonitrile in water).41 The eluted samples were used for mass spectrometry and Western blot analysis to identify the selected peptide-binding receptor. Confocal Imaging Colocalization of SD Peptide with Periostin. After incubation with FITC-SD peptide for 6 h, osteoblasts were fixed in 4% paraformaldehyde at room temperature for 15 min, followed by three PBS washes for 15 min. Subsequently, osteoblasts were incubated with permeabilization solution (PBS; 0.25% Triton X100) for 10 min, followed by three times cold PBS washes for 15 min. After blocking with blocking solution (PBS, 1% BSA, 0.1% Tween 20) for 1 h, osteoblasts were incubated with periostin antibodies (H-300, sc-67233) in blocking solution at 4 °C overnight and then washed with PBS 3 times. Then, osteoblasts were incubated with goat anti-rabbit secondary antibody for 1 h. Finally, nucleic acid was stained with DAPI for 10 min. Images were acquired on a confocal microscope (Zeiss LSM 700, Germany). Synthesis of PU. Polyurethane was synthesized in two steps: (1) preparation of polyurethane hard segments and (2) polymerization of hard segments and soft segments. The hard segments were obtained through a reaction between L-lysine diisocyanate (LDI, molecular weight 226) and N-methyldiethanolamine (MEDA, molecular weight 119.16) in acetone solution. The reactions were performed at 60 °C with constant stirring for 24 h to form an isocyanate terminated prepolymer in a closed reaction vessel. Afterward, the isocyanateterminated polyurethane prepolymer solutions were cooled to room temperature, and poly(ε-caprolactone) diol (PCL, molecular weight 1000), polyethylene glycol (PEG, molecular weight 2000), and acetone were added to the hard segments solution, initiating the polymerization reaction. The reactions continued at 60 °C with constant stirring for 72 h in a closed reaction vessel. Then, ethanol was added to the mixture and the reaction was kept at 60 °C with constant stirring for 48 h. After completion of the reaction, the reaction mixture was precipitated in petroleum ether to obtain the pure polyurethane from the mixture, and the final products were dried under in a vacuum oven at 25 °C. PU micelles were prepared by high-speed centrifugation and dialysis.42 First, deionized water was slowly added dropwise to 1% (w/v, PU/acetone) PU−acetone solution, and the solution was centrifuged at 15 000g for 10 min at 20 °C. Then, the supernatant was transferred to a dialysis bag (MWCO 3500) and dialyzed with deionized water for 1 day to remove organic solvent at room temperature. Finally, the PU micelle solution was filtered using a syringe filter with 0.22 μM pore size. Synthesis of SDSSD−PU. One milligram of SD-PEG-COOH was dissolved in deionized water and reacted with 1 mg of 1-(3(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC) and 1 mg ofN-hydroxysuccinimide (NHS) at 4 °C for 10 min. PU micelles were subsequently added to SD peptide solutions and the mixture was reacted overnight at 4 °C. Then, the reaction solution was purified by dialysis (MWCO = 3500) three times, washing with PBS (500 μL containing 10 μL of 0.5 M EDTA solution, pH 7.4). The pure SDSSD−PU was freeze-dried and stored at in low temperature.43 Preparation of siRNA/miRNA Formulations. The preparation of different siRNA/miRNA formulations was performed according to a previous study.44 PU or SDSSD−PU was diluted with ddH2O and sonicated for 10 min. Then, siRNA/miRNA solution was added to PU or SDSSD−PU solution and immediately vortexed at 4 °C. The prepared siRNA/miRNA formulations were placed under vortexed movement before using in all experiments. Characterization of SDSSD−PU. The average size and zeta potential of SDSSD−PU were detected by dynamic light scattering (DLS) using Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., U.K.).45 The morphology of SDSSD−PU was assessed with the Philips Tecnai 10 (Philips, Eindhoven, The Netherlands) transmission electron microscope (TEM) with an accelerating voltage of 100 kV.

Briefly, a drop of SDSSD−PU micelles solution was placed on a copper grid with Formvar film after negative staining with 1% (w/v) phosphor tungstic acid. After the liquid was blotted and air-dried, the particles morphology was measured by adjusting the parameters of the electron microscope. The loading capacity and serum stability of SDSSD−PU were assessed by agarose gel electrophoresis.46,47 SDSSD−PU-siRNA complexes with different SDSSD−PU/siRNA (w/w) ratios ranging from 1 to 20 were prepared. SDSSD−PU-siRNA complexes were mixed with 1 μL of 6× DNA gel loading buffer, and the mixture was loaded onto a 3% agarose gel (0.5 mg/mL ethidium bromide). The electrophoresis was run in 1× TAE buffer at 130 V for 30 min. Afterward, RNA was analyzed on an UV illuminator. For the serum stability assay, the free siRNA or SDSSD−PU-siRNA (N/P, 15:1 wt %) complexes were incubated with 50% fetal calf serum (FBS) for up to 24 h. According to the incubation times, aliquots from each sample were electrophoresed on a 2% agarose gel in the TAE buffer at 130 V for 30 min. Afterward, RNA was analyzed on an UV illuminator. Cell Uptake Assays of siRNA Formulations in Vitro. Briefly, PU-siRNA or SDSSD−PU-siRNA complexes prepared with FAMlabeled siRNA were incubated with cells at a density of 80%. After treatment for 6 h, the cells were washed three times with PBS and then visualized under a fluorescence microscope. Cytotoxicity Assays in Vitro. The Cell Titer-Blue Cell Viability Assay was performed to assess cell viability after cells were incubated with peptides, PU, or SDSSD−PU. Briefly, cells were exposed to the selected peptide, PU, or SDSSD−PU at different concentrations. After treatment for 12 h, the cell viability was detected by Cell Titer-Blue Cell Viability Assay according to the manufacturer’s protocol.48 Toxicity Assays in Vivo. Mice were injected with 200 μL of saline, PU, or SDSSD−PU via tail vein injection (PU and SDSSD−PU were dissolved in 200 μL of RNase/DNase-free saline at a dose of 10 mg/ kg). After administration for 24 h, heart, liver, spleen, and kidney were collected for H&E staining analysis. Biochemical parameters, i.e., ALT, AST, CK-MB, and BUN, were analyzed using a clinical chemistry analyzer (Cruinn Diagnostics Ltd., Ireland). Levels of serum IL-6, TNF-α and IFN-α were determined with an ELISA using ELISA kit (eBioscience, USA).49,50 Tissue Distribution of siRNA in Vivo. Mice were divided into three groups (saline, PU-siRNA, and SDSSD−PU-siRNA). Mice in the saline group were injected with 200 μL of saline via tail vein, while the remaining groups received the FAM-labeled siRNA delivery system at a dose of 10 mg/kg with different nanoparticle formulations at an equivalent volume. Twelve hours later, all the mice were sacrificed and the major organs (long bones, heart, lung, and liver) were collected from each mouse for detection of the fluorescence signal using MI SE software (Bruker, Germany). The concentration of SDSSD−PU is 1 mg/mL, and 0.2 mL/(mice/injection). Bioluminescence Imaging of Mice. Six 6-week-old mice were randomly divided into three groups, and injected with saline, PUsiRNA, and SDSSD−PU-siRNA. The concentration of SDSSD−PU was 1 mg/mL, and we used 0.2 mL/(mice/injection). The siRNA concentration for mouse injection was 10 mg/kg. Mice were sacrificed 12 h after injection. Tissue fixation was performed using 4% paraformaldehyde (PFA). Long bones, brain, heart, lung, and liver were collected and tested for delivery efficiency and specificity. Bioluminescence imaging of mice was carried out using a LB983 fluorescence imaging system (Berthold Night Owl, Germany). The parameter set is excitation light 480 nm, emitted light 520 nm. Cell-Selective Delivery in Vivo. Femurs from the three groups were decalcified with 10% EDTA for one month. After dehydration, femur tissue was embedded in paraffin and 4 μM sections were prepared for staining. Cell-selective delivery efficiency was evaluated by histological analysis. Green florescence signals indicating SDSSD− PU localizations were observed by florescence microscope (Nikon, Japan). An anti-DMP1 monoclonal antibody 8G10.3 at a 1:500 dilution (Gift from Dr. Chunlin Qin) was used for labeling osteoblasts. Goat-anti-mouse H&L secondary antibody was used at a 1:1000 dilution (Abcam, USA). DAPI (5%, Sigma, USA) was used for localization of the cell nucleus. 5765

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ACS Nano Gene Knockdown in GFP Transgenic Mice. GFP transgenic mice were divided into two groups. One group was injected with GFP siRNA in 200 μLof SDSSD−PU solution (at a siRNA dose of 10 mg/ kg), while another group was injected with SDSSD−PU-control siRNA at an equivalent volume. Briefly, for tissue-selective gene knockdown analysis, the major organs, including bone (femur and tibia), brain, heart, liver, and lung, were collected for fluorescence signal detection by MI SE software (Bruker, Germany) after injection for 12 h. Total RNA and protein from organs were extracted 12 h after the injection via tail vein and analyzed by real-time PCR and Western blot, respectively. In addition, the femur and tibia from each group were obtained for analyzing the GFP fluorescence signals on the boneformation surfaces. For cell-selective gene knockdown analysis, the levels of GFP mRNA harvested from osteoblasts were analyzed by realtime PCR and the levels of GFP protein harvested from osteoblasts were analyzed by Western blot. FACS Isolation of Osteoblasts from Mice. Bone marrow stromal cells were collected from the femurs and tibiae of GFP transgenic mice and OVX mice. PerCP-labeled antibody to mouse ALP (R&D systems, AAST0108101, 1:50) was used for FACS to isolate osteoblasts. After a wash with PBS and 1% BSA, the cells were directly stained with the PerCP-labeled antibody to ALP. Then, stained cell populations were washed three times for FACS analysis. The selected ALP+ cells were used for real-time PCR analysis and Western blot analysis. Therapeutic Evaluation of Anti-miR-214 Delivered by SDSSD−PU to OVX Mice. For antiosteoporosis analysis, 18 OVX mice were divided into three groups. The OVX control group was untreated, the OVX + anti-scramble group was treated with 200 μL of SDSSD−PU-anti-scramble, and the OVX + anti-miR-214 group was treated with 200 μL of SDSSD−PU-anti-miR-214, with a siRNA dose of 10 mg/kg at an interval of 1 week. All mice were injected with calcein green (10 mg/kg) before drug administration and in the 2 days before sacrifice for double labeling of the bone formation. After drug administration for one month, all mice were sacrificed. The levels of miR-214 in osteoblasts sorted from all mice were detected by real-time PCR. Proximal tibias were collected to demonstrate the new bone formation by confocal microscopy and to show the three-dimensional trabecular architecture by micro-CT system. Bone mineral density of all mice was calculated by the micro-CT system, and the mineral apposition rate was determined by bone dynamic histomorphometric analysis. Western Blot Analysis. Tissues or cells were lysed in lysis buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 0.1% SDS, 2 mM dithiothreitol (DTT), 0.5% NP-40, 1 mM PMSF and protease inhibitor cocktail) on ice for 30 min. Protein fractions were collected by centrifugation at 15 000g at 4 °C for 10 min. The protein samples were subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% BSA and incubated with specific antibodies overnight. A horseradish peroxidase labeled secondary antibody was added and visualized using an enhanced chemiluminescence kit (Pierce, USA). We used periostin antibody (Santa Cruz, CA) to examine periostin and anti-GFP (MO48-3; Sigma, USA) to examine GFP protein in osteoblasts. Micro-CT Analysis. For the distal femur, the whole secondary spongiosa of the left distal femur from each mouse was scanned ex vivo using a micro-CT system (μCT35, Scanco Medical, Switzerland). Briefly, 423 slices with a voxel size of 10 μM were scanned at the region of the distal femur beginning at the growth plate and extending proximally along the femur diaphysis. Eighty continuous slices beginning at 0.1 mm from the most proximal aspect of the growth plate in which both condyles were no longer visible were selected for analysis. For the lumbar vertebral (LV) bodies, the entire region of secondary spongiosa between the proximal and distal aspects from LV5 was scanned by micro-CT with a voxel size of 10 μm. Three hundred and fifty slices, which included the entire region of the LV5 body, were analyzed. All trabecular bone from each selected slice was segmented for three-dimensional reconstruction (sigma = 1.2, supports = 2, and threshold = 180) to calculate the BMD.51

Bone Histomorphometric Analysis. The distal femur was dehydrated in graded concentrations of ethanol and embedded without decalcification in modified methyl methacrylate (MMA) using our previously established protocol after micro-CT analysis. Frontal sections of trabecular bone were obtained from the distal femur or LV5 body at a thickness of 15 μM with a Leica SM2500E microtome (Leica Microsystems, Germany). Bone dynamic histomorphometric analysis for MAR was performed using professional image analysis software (ImageJ, NIH, USA) under fluorescence microscopy (Leica image analysis system, Q500MC). Statistical Analyses. All numerical data are expressed as the mean ± SD. Significant differences among groups were analyzed by one-way analysis of variance with a post-hoc test to determine group differences in the study parameters. All statistical analyses were performed with SPSS software, version 16.0. Significant differences between two groups were determined by Student’s t test. P < 0.05 was considered statistically significant.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.5b07828. Results of cytotoxicity assays and protein binding of SDSSD peptides in vitro, characterization and cytotoxicity assays of SDSSD−PU (PDF) Table S1 (XLSX) Table S2 (XLSX)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Author Contributions #

All these authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank Dr. Yingxian Li (China Astronaut Research and Training Center) for valuable advice. Appreciate Dr. Guosong Chen (FuDan University) for the help of biochemistry analysis and appreciate for the Antibodies and help from Dr. Chunlin Qin (Baylor college of Dentistry). This work was supported by The Fundamental Research Funds for the Central Universities (21614608; 11615206;TJ1504219036; 21615480); National Natural Science Foundation Projects (81370971, 31101251, 81271110, 81300840, 81470715 and 81300908) Guangdong Natural Science Funds (2014A030313358); Science and Technology Major Project of Guangdong Province (2015B020225005) and Guangdong Natural Science Funds for Distinguished Young Scholar (S2013050013880); XYQ2013080; Education Ministry’s New Century Excellent Talents Supporting Plan NCET-13−0426; 2012 Recruitment Program of Global Experts-1000 Plan (YS) and National Science-technology Support Plan Projects 2014BAI04B07. REFERENCES (1) Sims, N. A.; Martin, T. J. Coupling The Activities of Bone Formation and Resorption: A Multitude of Signals within the Basic Multicellular Unit. BoneKEy Rep. 2014, 3, 481. (2) Rachner, T. D.; Khosla, S.; Hofbauer, L. C. Osteoporosis: Now and the Future. Lancet 2011, 377, 1276−1287. 5766

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ACS Nano (3) Canalis, E. New Treatment Modalities in Osteoporosis. Endocr Pract. 2010, 16, 855−863. (4) Daddona, P. E.; Matriano, J. A.; Mandema, J.; Maa, Y. F. Parathyroid Hormone (1−34)-Coated Microneedle Patch System: Clinical Pharmacokinetics and Pharmacodynamics for Treatment of Osteoporosis. Pharm. Res. 2011, 28, 159−165. (5) Delmas, P. D.; Ensrud, K. E.; Adachi, J. D.; Harper, K. D.; Sarkar, S.; Gennari, C.; Reginster, J. Y.; Pols, H. A.; Recker, R. R.; Harris, S. T.; Wu, W.; Genant, H. K.; et al. Efficacy of Raloxifene on Vertebral Fracture Risk Reduction in Postmenopausal Women with Osteoporosis: Four-Year Results from a Randomized Clinical Trial. J. Clin. Endocrinol. Metab. 2002, 87, 3609−3617. (6) Black, D. M.; Delmas, P. D.; Eastell, R.; Reid, I. R.; Boonen, S.; Cauley, J. A.; Cosman, F.; Lakatos, P.; Leung, P. C.; Man, Z.; et al. R. Once-yearly Zoledronic Acid for Treatment of Postmenopausal Osteoporosis. N. Engl. J. Med. 2007, 356, 1809−1822. (7) Yin, H.; Kanasty, R. L.; Eltoukhy, A. A.; Vegas, A. J.; Dorkin, J. R.; Anderson, D. G. Non-viral Vectors for Gene-Based Therapy. Nat. Rev. Genet. 2014, 15, 541−555. (8) Mingozzi, F.; High, K. A. Therapeutic in Vivo Gene Transfer for Genetic Disease Using AAV: Progress and Challenges. Nat. Rev. Genet. 2011, 12, 341−355. (9) Bessis, N.; GarciaCozar, F. J.; Boissier, M. C. Immune Responses to Gene Therapy Vectors: Influence on Vector Function and Effector Mechanisms. Gene Ther. 2004, 11, S10−S17. (10) Waehler, R.; Russell, S. J.; Curiel, D. T. Engineering Targeted Viral Vectors for Gene Therapy. Nat. Rev. Genet. 2007, 8, 573−587. (11) Kanasty, R.; Dorkin, J. R.; Vegas, A.; Anderson, D. Delivery Materials for siRNA Therapeutics. Nat. Mater. 2013, 12, 967−977. (12) Field, L. D.; Delehanty, J. B.; Chen, Y.; Medintz, I. L. Peptides for Specifically Targeting Nanoparticles to Cellular Organelles: Quo Vadis. Acc. Chem. Res. 2015, 48, 1380−1390. (13) Hilgenbrink, A. R.; Low, P. S. Folate Receptor-Mediated Drug Targeting: from Therapeutics to Diagnostics. J. Pharm. Sci. 2005, 94, 2135−2146. (14) Wijagkanalan, W.; Kawakami, S.; Hashida, M. Glycosylated Carriers for Cell-Selective and Nuclear Delivery of Nucleic Acids. Front. Biosci., Landmark Ed. 2011, 16, 2970−2987. (15) Weinberg, W.; Frazier-Jessen, M.; Wu, W.; Weir, A.; Hartsough, M.; Keegan, P.; Fuchs, C. Development and Regulation of Monoclonal Antibody Products: Challenges and Opportunities. Cancer Metastasis Rev. 2005, 24, 569−584. (16) Wu, Z.; Tang, L.-J.; Zhang, X.-B.; Jiang, J.-H.; Tan, W. AptamerModified Nanodrug Delivery Systems. ACS Nano 2011, 5, 7696− 7699. (17) Kamaly, N.; Xiao, Z.; Valencia, P. M.; Radovic-Moreno, A. F.; Farokhzad, O. C. Targeted Polymeric Therapeutic Nanoparticles: Design, Development and Clinical Translation. Chem. Soc. Rev. 2012, 41, 2971−3010. (18) Hobbs, S. K.; Monsky, W. L.; Yuan, F.; Roberts, W. G.; Griffith, L.; Torchilin, V. P.; Jain, R. K. Regulation of Transport Pathways in Tumor Vessels: Role of Tumor Type and Microenvironment. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 4607−4612. (19) Blanco, E.; Shen, H.; Ferrari, M. Principles of Nanoparticle Design for Overcoming Biological Barriers to Drug Delivery. Nat. Biotechnol. 2015, 33, 941−951. (20) Low, S. A.; Kopeček, J. Targeting Polymer Therapeutics to Bone. Adv. Drug Delivery Rev. 2012, 64, 1189−1204. (21) Wang, D.; Miller, S. C.; Kopečková, P.; Kopeček, J. BoneTargeting Macromolecular Therapeutics. Adv. Drug Delivery Rev. 2005, 57, 1049−1076. (22) Neale, J. R.; Richter, N. B.; Merten, K. E.; Grant Taylor, K.; Singh, S.; Waite, L. C.; Emery, N. K.; Smith, N. B.; Cai, J.; Pierce, W. M., Jr Bone Selective Effect of an Estradiol Conjugate with a Novel Tetracycline-Derived Bone-Targeting Agent. Bioorg. Med. Chem. Lett. 2009, 19, 680−683. (23) Hirabayashi, H.; Fujisaki, J. Bone-Specific Drug Delivery Systems. Clin. Pharmacokinet. 2003, 42, 1319−1330.

(24) Sekido, T.; Sakura, N.; Higashi, Y.; Miya, K.; Nitta, Y.; Nomura, M.; Sawanishi, H.; Morito, K.; Masamune, Y.; Kasugai, S.; et al. Novel Drug Delivery System to Bone Using Acidic Oligopeptide: Pharmacokinetic Characteristics and Pharmacological Potential. J. Drug Target. 2001, 9, 111−121. (25) Kasugai, S.; Fujisawa, R.; Waki, Y.; Miyamoto, K.-I.; Ohya, K. Selective Drug Delivery System to Bone: Small Peptide (Asp)6 Conjugation. J. Bone Miner. Res. 2000, 15, 936−943. (26) Yarbrough, D.; Hagerman, E.; Eckert, R.; He, J.; Choi, H.; Cao, N.; Le, K.; Hedger, J.; Qi, F.; Anderson, M.; et al. Specific Binding and Mineralization of Calcified Surfaces by Small Peptides. Calcif. Tissue Int. 2010, 86, 58−66. (27) Liang, C.; Guo, B.; Wu, H.; Shao, N.; Li, D.; Liu, J.; Dang, L.; Wang, C.; Li, H.; Li, S.; et al. Aptamer-Functionalized Lipid Nanoparticles Targeting Osteoblasts as a Novel RNA InterferenceBased Bone Anabolic Strategy. Nat. Med. 2015, 21, 288−294. (28) Perrin, D. D. Binding of Tetracyclines to Bone. Nature 1965, 208, 787−788. (29) Kay, M. I.; Young, R. A.; Posner, A. S. Crystal Structure of Hydroxyapatite. Nature 1964, 204, 1050−1052. (30) Albert, A.; Rees, C. W. Avidity of the Tetracyclines for the Cations of Metals. Nature 1956, 177, 433−434. (31) Russell, R. G.; Watts, N. B.; Ebetino, F. H.; Rogers, M. J. Mechanisms of Action of Bisphosphonates: Similarities and Differences and Their Potential Influence on Clinical Efficacy. Osteoporosis Int. 2008, 19, 733−759. (32) Wang, D.; Miller, S. C.; Shlyakhtenko, L. S.; Portillo, A. M.; Liu, X.-M.; Papangkorn, K.; Kopečková, P.; Lyubchenko, Y.; Higuchi, W. I.; Kopeček, J. Osteotropic Peptide That Differentiates Functional Domains of the Skeleton. Bioconjugate Chem. 2007, 18, 1375−1378. (33) Favus, M. J. Bisphosphonates for Osteoporosis. N. Engl. J. Med. 2010, 363, 2027−2035. (34) Zhang, G.; Guo, B. S.; Wu, H.; Tang, T.; Zhang, B. T.; Zheng, L. Z.; He, Y. X.; Yang, Z. J.; Pan, X. H.; Chow, H.; et al. A Delivery System Targeting Bone Formation Surfaces to Facilitate Rnai-Based Anabolic Therapy. Nat. Med. 2012, 18, 307−314. (35) Mallikaratchy, P. R.; Ruggiero, A.; Gardner, J. R.; Kuryavyi, V.; Maguire, W. F.; Heaney, M. L.; McDevitt, M. R.; Patel, D. J.; Scheinberg, D. A. A Multivalent DNA Aptamer Specific for the B-Cell Receptor on Human Lymphoma and Leukemia. Nucleic Acids Res. 2011, 39, 2458−2469. (36) Horiuchi, K.; Amizuka, N.; Takeshita, S.; Takamatsu, H.; Katsuura, M.; Ozawa, H.; Toyama, Y.; Bonewald, L. F.; Kudo, A. Identification and Characterization of a Novel Protein, Periostin, with Restricted Expression to Periosteum and Periodontal Ligament and Increased Expression by Transforming Growth Factor β. J. Bone Miner. Res. 1999, 14, 1239−1249. (37) McGuire, M. J.; Li, S.; Brown, K. C. Biopanning of Phage Displayed Peptide Libraries for the Isolation of Cell-Specific Ligands. Methods Mol. Biol. (Clifton, N.J.) 2009, 504, 291−321. (38) McGuire, M. J.; Gray, B. P.; Li, S.; Cupka, D.; Byers, L. A.; Wu, L.; Rezaie, S.; Liu, Y. H.; Pattisapu, N.; Issac, J.; et al. Identification and Characterization of a Suite of Tumor Targeting Peptides for NonSmall Cell Lung Cancer. Sci. Rep. 2014, 4, 4480. (39) Hofmeister, L. H.; Lee, S. H.; Norlander, A. E.; Montaniel, K. R.; Chen, W.; Harrison, D. G.; Sung, H. J. Phage-Display-Guided Nanocarrier Targeting to Atheroprone Vasculature. ACS Nano 2015, 9 (4), 4435−4446. (40) Teranishi, Y.; Hur, J. Y.; Welander, H.; Frånberg, J.; Aoki, M.; Winblad, B.; Frykman, S.; Tjernberg, L. O. Affinity Pulldown of γSecretase and Associated Proteins from Human and Rat Brain. J. Cell Mol. Med. 2010, 14, 2675−2686. (41) Schiapparelli, L. M.; McClatchy, D. B.; Liu, H. H.; Sharma, P.; Yates, J. R.; Cline, H. T. Direct Detection of Biotinylated Proteins by Mass Spectrometry. J. Proteome Res. 2014, 13, 3966−3978. (42) Mahanta, A. K.; Mittal, V.; Singh, N.; Dash, D.; Malik, S.; Kumar, M.; Maiti, P. Polyurethane-Grafted Chitosan as New Biomaterials for Controlled Drug Delivery. Macromolecules 2015, 48, 2654−2666. 5767

DOI: 10.1021/acsnano.5b07828 ACS Nano 2016, 10, 5759−5768

Article

ACS Nano (43) Wu, C.; Gong, F.; Pang, P.; Shen, M.; Zhu, K.; Cheng, D.; Liu, Z.; Shan, H. An RGD-Modified MRI-Visible Polymeric Vector for Targeted siRNA Delivery to Hepatocellular Carcinoma In Nude Mice. PLoS One 2013, 8, e66416. (44) Choi, K. Y.; Silvestre, O. F.; Huang, X.; Hida, N.; Liu, G.; Ho, D. N.; Lee, S.; Lee, S. W.; Hong, J. I.; Chen, X. A Nanoparticle Formula for Delivering siRNA or miRNAs to Tumor Cells in Cell Culture and in Vivo. Nat. Protoc. 2014, 9, 1900−1915. (45) Ding, M.; Song, N.; He, X.; Li, J.; Zhou, L.; Tan, H.; Fu, Q.; Gu, Q. Toward the Next-Generation Nanomedicines: Design of Multifunctional Multiblock Polyurethanes for Effective Cancer Treatment. ACS Nano 2013, 7, 1918−1928. (46) Su, S.; Tian, Y.; Li, Y.; Ding, Y.; Ji, T.; Wu, M.; Wu, Y.; Nie, G. ″Triple-Punch″ Strategy for Triple Negative Breast Cancer Therapy with Minimized Drug Dosage and Improved Antitumor Efficacy. ACS Nano 2015, 9, 1367−1378. (47) Khatri, N.; Baradia, D.; Vhora, I.; Rathi, M.; Misra, A. cRGD Grafted Liposomes Containing Inorganic Nano-Precipitate Complexed siRNA for Intracellular Delivery in Cancer Cells. J. Controlled Release 2014, 182, 45−57. (48) O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Investigation of the Alamar Blue (resazurin) Fluorescent Dye for the Assessment of Mammalian Cell Cytotoxicity. Eur. J. Biochem. 2000, 267, 5421−5426. (49) Semple, S. C.; Akinc, A.; Chen, J.; Sandhu, A. P.; Mui, B. L.; Cho, C. K.; Sah, D. W.; Stebbing, D.; Crosley, E. J.; Yaworski, E.; et al. Rational Design of Cationic Lipids for siRNA Delivery. Nat. Biotechnol. 2010, 28, 172−176. (50) Zhou, H. F.; Yan, H.; Pan, H.; Hou, K. K.; Akk, A.; Springer, L. E.; Hu, Y.; Allen, J. S.; Wickline, S. A.; Pham, C. T. Peptide-siRNA Nanocomplexes Targeting NF-κB Subunit p65 Suppress Nascent Experimental Arthritis. J. Clin. Invest. 2014, 124 (10), 4363−4374. (51) Iwaniec, U. T.; Wronski, T. J.; Liu, J.; Rivera, M. F.; Arzaga, R. R.; Hansen, G.; Brommage, R. PTH Stimulates Bone Formation in Mice Deficient in Lrp5. J. Bone Miner. Res. 2007, 22, 394−402.

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