Engineering of Bone- and CD44-Dual-Targeting Redox-Sensitive

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Applications of Polymer, Composite, and Coating Materials

Engineering of Bone- and CD44-Dual-Targeting Redox-Sensitive Liposomes for the Treatment of Orthotopic Osteosarcoma Shuaishuai Feng, Zi-Xin Wu, Ziyan Zhao, Jinhu Liu, Kaoxiang Sun, Chuanyou Guo, Hongbo Wang, and Zimei Wu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b18820 • Publication Date (Web): 25 Jan 2019 Downloaded from http://pubs.acs.org on January 27, 2019

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ACS Applied Materials & Interfaces

Engineering of Bone- and CD44-Dual-Targeting Redox-Sensitive Liposomes for the Treatment of Orthotopic Osteosarcoma Shuaishuai Fenga, Zi-Xin Wub, Ziyan Zhaoa, Jinhu Liua, Kaoxiang Suna, Chuanyou Guob, Hongbo Wanga, Zimei Wua, c* aSchool

of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai

University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in University of Shandong, Yantai University, Yantai 264005, PR China bQingdao

cSchool

Municipal Hospital, Qingdao 266071, Shandong Province, PR China

of Pharmacy, University of Auckland, Auckland 1142, New Zealand

ACS Paragon Plus Environment

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ABSTRACT

This study aimed to develop an efficient step-by-step osteosarcoma (OS)-targeting liposome system functionalized with a redox-cleavable, bone- and cluster of differentiation 44 (CD44)dual-targeting polymer. Furthermore, the effect of coadministration of a tumor-penetrating peptide, internalizing-RGD (iRGD), was investigated. First, a bone-targeting moiety, alendronate (ALN), was conjugated with hyaluronic acid (HA), a ligand for CD44. This ALN-HA conjugate was coupled with DSPE-PEG2000-COOH through a bioreducible disulfide linker (-SS-) to obtain a functionalized lipid, ALN-HA-SS-L, to be post-inserted into preformed liposomes loaded with doxorubicin (DOX). The roles of ALN, HA and the redox-sensitivity of the ALN-HA-SS-L liposomes (ALN-HA-SS-L-L) in the anti-osteosarcoma effect were critically evaluated against various reference liposomal formulations (with only ALN, HA or redox sensitivity). ALN-HASS-L-L displayed a zeta potential of -26.07±0.32 mV and selectively disassembled in the presence of a reducing agent, 10 mM glutathione (GSH), which can be found in cancer cells. Compared to various reference liposomes, ALN-HA-SS-L-L/DOX had significantly higher cytotoxicity to human OS MG-63 cells alongside high and rapid cellular uptake. In the orthotopic OS nude mouse models, ALN-HA-SS-L-L/DOX showed remarkable tumor growth suppression and prolonged survival time. This result was further improved by the coadministration of iRGD. The antitumor effects of various liposomes were ranked in the same order as the degree of tumor biodistribution shown by in vivo/ex vivo imaging: ALN-HA-SS-L-L coadministered with iRGD > ALN-HA-SS-L-L > HA-SS-L-L > HA-L-L > PEG-L> free drug. ALN-HA-SS-L-L/DOX also reduced the cardiotoxicity of DOX and lung metastases. Overall, this study demonstrated that ALN-HA-SS-L-L/DOX, equipped with bone- and CD44-dual-


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targeting abilities and redox sensitivity, could be a promising OS-targeted therapy. The efficacy could also be augmented by coadministration of iRGD.

KEYWORDS Osteosarcoma (OS); Alendronate (ALN); Hydroxyapatite; Hyaluronic acid (HA); Redoxsensitive liposomes; iRGD

1. INTRODUCTION Osteosarcoma (OS), is the most common primary malignant bone tumor, and mainly affects children and adolescents.1,2 At present, chemotherapy (pre- and postoperative), usually with multiple chemotherapeutic agents, remains the gold standard for the treatment of OS; however, it results in severe side effects in patients. Despite advances in cancer research, the long-term survival of patients with OS is low,3,4 and most patients die of lung metastases.2 Therefore, innovative and effective therapeutic approaches are urgently needed.2 Liposomes have been widely used as drug carriers because of their advantages in biocompatibility and flexibility with both hydrophilic and hydrophobic drug loading.5 To prevent rapid uptake and accumulation by phagocytic cells of the reticuloendothelial system (RES),6 surface modification with a hydrophilic polymer, polyethylene glycol (known as PEGylation, for example, liposomal doxorubicin, Doxil7) has been the gold standard approach. To further improve the efficacy, tumor environmental stimuli-triggered drug release has been highly demanded in cancer treatment in recent years. For example, redox-sensitive nanoparticles, typically containing disulfide bonds (-SS-), have been employed to achieve burst release of encapsulated drugs in response to the intracellular redox potential of tumors.8-10 The disulfide


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bonds are stable during blood circulation and in the extracellular milieu but are prone to rapid cleavage (from minutes to hours) through thiol-disulfide exchange reactions with intracellular reducing molecules, especially glutathione (GSH).11,12 GSH presents much higher concentrations inside cells (0.5-10 mM) than in the extracellular milieu (2-20 μM).13-15 Furthermore, the concentration of GSH in tumor tissues is at least 4-fold that in normal tissues.16 This large discrepancy is the premise of designing redox-sensitive liposomes for tumor-targeted intracellular drug delivery.9,10,17 Another widely used approach to improve the anticancer efficacy of nanoparticles, including liposomes, is through receptor-mediated cancer cell uptake. Cluster of differentiation 44 (CD44), a cell-surface receptor highly expressed on MG-63 cells,18,19 can be exploited for targeted chemotherapy of OS. CD44 can recognize and bind hyaluronic acid (HA),20 a biocompatible native anionic glycosaminoglycan with the potential to be chemically modified.21,22 Although CD44 is also expressed in normal epithelial cells and HA is part of the matrix of normal tissues, HA-CD44 interactions have recently been exploited for tumor-targeted drug delivery, given that these nanocarriers preferentially transport into tumor tissue by exploiting the leaky vasculature of solid tumors.23,24 Previously, we explored the use of redox-sensitive and CD44-targeted liposomes for the treatment of OS in which liposomes were stabilized with GSH-cleavable PEG coating and functionalized by electrostatic HA coating.8 Compared with non-redox-sensitive or non-CD44-targeted liposomes, particularly the drug solution (doxorubicin, DOX), the dualfunction liposomes demonstrated specific intracellular drug delivery ability to OS, which led to significant inhibitory effects on tumor growth in subcutaneously implanted MG-63 xenograft mouse models.8 It was considered that these liposomes could accumulate in tumor tissue and then undergo cellular uptake via HA-CD44 interactions, subsequently releasing their payload


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into the cytoplasm owing to their redox sensitivity. We envisaged that further improvement could be achieved by conferring liposomes with additional bone-binding ability. Hydroxyapatite (Ca10(PO4)6(OH)2) is a main mineral component of bone. Bone diseases, including OS,25 can cause local inflammation and/or result in exposure of HA to blood. Thus, hydroxyapatite can be made an attractive binding target through the use of bisphosphonates, such as alendronate (ALN),26 via chelation with the Ca2+ of hydroxyapatite. Studies have demonstrated preferential attachment of ALN to the exposed hydroxyapatite, achieving bone resorption.27 Recently, this specific affinity has been exploited in the design of drug-polymer conjugates for the treatment of osteoporosis or bone cancer.28-30 In this work, we aimed to develop a bone-targeting and redox-sensitive liposomal system based on previous work.8 A polymer, ALN-HA-SS-L, was synthesized with ALN and HA as bone- and OS cell-targeting moieties that were conjugated with PEG2000-DSPE via cystamine (containing a -SS- bond) for tumor-targeted intracellular delivery of DOX as a model anticancer drug. Non-bone-targeting polymers with and without redox sensitivity were also synthesized for comparison. It was hypothesized that the ALN-HA-SS-L-functionalized liposomes (ALN-HASS-L-L) could improve antitumor efficacy and reduce toxicity by exploiting a step-by-step targeting approach. First, ALN-HA-SS-L-L would selectively bind to the exposed hydroxyapatite of the inflammatory bone structure. Second, at the OS site, HA would promote CD44 receptor-mediated endocytosis by OS cells. Third, once the liposomes were internalized by tumor cells, intracellular GSH would promote polymer shedding from the liposomes by the cleavage of the –SS- bonds, allowing drug release from the plain liposomes. In vitro OS MG-63 cell culture and in vivo tumor models were used to evaluate the OS-targeting effect. Instead of


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using subcutaneously implanted xenograft models, an orthotopic tumor model was established, as the latter type of model may be more suitable for clinical translation.31,32 In addition, given that OS is a bone cancer sarcoma, the dense extracellular matrix of OS cells restricts nanoparticle penetration into tumors,33 limiting the enhanced permeability and retention (EPR) effect. Therefore, selectively increasing the permeability of OS tissues is essential to augment OS treatment with liposomes. Internalizing-RGD (iRGD), a cyclic peptide with an amino acid sequence of CRGDK/RGPD/EC, has been shown to enhance intratumoral drug accumulation when it is conjugated to or coadministered with anticancer drugs or delivery carriers.34,35 The iRGD peptide homes to tumors through three steps: the RGD motif mediates binding to αv integrins on the tumor endothelium, where it undergoes proteolytic cleavage resulting in exposure of the binding motif for neuropilin-1 (NRP-1), which mediates penetration into tissue and cells.34 The αv integrins are also reported to be highly expressed in cancer cells but less in normal cells36,37 and, in the case of OS, are weakly expressed in osteoblasts but strongly expressed in osteoclasts.38,39 High expression of αv40 and NRP-141 is often observed at the primary site of OS. Coadministration of iRGD has been shown to selectively enhance the vascular and tissue permeability of tumors overexpressing αv integrins and NRP-142 and thus may provide additional benefit in treating OS with liposomes. 2. EXPERIMENTAL SECTION 2.1. Materials Alendronate sodium (ALN) was obtained from J&K Scientific LTD. HA (molecular weight 20-40 kDa) was purchased from Shandong Freda Biopharmaceutical CO., LTD (Jinan, China). Cystamine dihydrochloride (CYS• 2HCl) and 1,6-hexanediamine (HMDA) were purchased from Adamas Reagent Co., Ltd. DSPE-mPEG2000 was purchased from Shanghai Advanced Vehicle


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Technology Pharmaceutical Ltd. (Shanghai, China), while DSPE-PEG2000-COOH (MW 2900) was purchased from Xi’an Ruixi Biological Technology Co., Ltd (Xi’an, China). Doxorubicin hydrochloride (DOX•HCl), soybean phosphatidylcholine (SPC), cholesterol, Nhydroxysulfosuccinimide sodium salt (sulfo-NHS), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC•HCl), glutathione (GSH) and various dialysis bags (MWCO 3500 Da, 7000 Da or 10 kDa) were all purchased from Aladdin (Shanghai, China). For cell culture studies, the human osteosarcoma (OS) MG-63 cell line was purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Minimum essential medium (MEM), MEM nonessential amino acid solution, sodium pyruvate (100 mM) and fetal bovine serum (FBS) were purchased from Gibco (Shanghai, China). Hoechst 33342 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were from Sigma-Aldrich (St. Louis, Mo, USA). For animal studies, female BALB/c nude mice (18~20 g) were purchased from Beijing Vital River Laboratory Animals Technology Co., Ltd. (Beijing, China). The tumor model construction and efficacy study were carried out at Yantai Raphael Biotechnology Co., Ltd. Care and handling of animals, with ethical approval, was performed according to the guidelines of the Experimental Animals Administrative Committee of Yantai University. 2.2. Synthesis of functionalized polymers To introduce various functions into liposomes by surface coating, various polymers were synthesized and conjugated with the phospholipid DSPE-PEG2000 at the distal end of the PEG chain (Figure 1).


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Figure 1. Synthetic schemes of intermediate products A) CYS-HA-ALN, B) CSY-HA (redox sensitive), C) redox-insensitive HMDA-HA, and the final product D) bone- and CD44-dualtargeting and redox-sensitive lipid ALN-HA-SS-L. 2.2.1. Synthesis of intermediate polymers First, ALN was conjugated with HA using NHS and EDC as coupling agents43,44 (Figure 1A). Briefly, HA (300 mg, equivalent to 0.75 mmol D-glucuronic acid/N-acetyl-D-glucosamine units) was dissolved in 60 ml of water with NHS (103 mg, 0.9 mmol) and EDC (171 mg, 0.9 mmol). The mixture was stirred for 1 h to activate the carboxylic acid moiety. Alendronate sodium (102


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mg, 0.375 mmol) was added to the reaction mixture and kept at room temperature under stirring for 24 h. The product was purified using dialysis tubes (MWCO 3500 Da) against water for 48 h. The purified HA-ALN solution was lyophilized, and the dry product was stored at 4 °C until further use. To confer redox sensitivity, CYS was used to introduce a -SS- bond into HA-ALN, forming CYS-HA-ALN (Figure 1A) through the reaction of the carboxylic acid groups of HA with the amino group of CYS.45,46 In brief, HA-ALN (200 mg, 0.306 mmol) was dissolved in 40 ml of PBS (0.01 M, pH 7.4). After the addition of sulfo-NHS and EDC (molar ratio of HA:sulfoNHS:EDC = 1:2:2), the mixture was stirred for 15 min to activate the carboxyl groups of HA. Then, CYS (344.25 mg, 1.53 mmol) was added dropwise, and the mixture was kept for 24 h at room temperature under stirring to allow the reaction to proceed. The resulting solution was dialyzed (MWCO 3500 Da) against deionized water for 24 h to remove the unreacted CYS, sulfo-NHS and EDC. Finally, the solution containing CYS-HA-ALN was lyophilized and stored at 4 °C until further use. Using the above reaction conditions, CYS (1120 mg, 5 mmol) was added to the HA solution (200 mg, 0.5 mmol) to obtain CYS-HA (Figure 1B). Similarly, a non-redox-sensitive HMDAHA conjugate was prepared (Figure 1C) as described for CYS-HA with HMDA replacing CYS. 2.2.2. Synthesis of functionalized lipids to be post-inserted into liposomes Finally, amphiphilic ALN-HA-SS-L, HA-SS-L and HA-L to be post-inserted into liposomes were prepared by conjugation of DSPE-PEG2000-COOH with CYS-HA-ALN, CYS-HA and HMDA-HA, respectively, all through amide bond formation,47 as shown for ALN-HA-SS-L (Figure 1D).


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First, DSPE-PEG2000-COOH was dissolved in water, and its carboxyl group was activated with NHS and EDC (molar ratios 1:2:2) at room temperature for 2 h to form DSPE-PEG2000-COOsuccinimide. CYS-HA-ALN was added dropwise (molar ratio 1.5:1), mixed, and allowed to react for 24 h. The mixture was dialyzed (MWCO 7000 Da) against water for 24 h to remove unreacted materials. After freeze-drying, the final product containing ALN-HA-SS-L was obtained as a white powder and stored at 4 °C until further use. The syntheses of HA-SS-L and HA-L were similar to those described above, except that CYSHA-ALN was replaced with CYS-HA and HMDA-HA, respectively. 2.3. Preparation of liposomes Initially, plain liposomes were prepared using the thin-film hydration method.48 SPC and cholesterol at a molar ratio of 3:2 were dissolved in chloroform/methanol (3:1, v/v) and dried using a rotary evaporator to obtain a thin lipid film. Following removal of residual solvent under vacuum, the thin film was hydrated with a 200 mM ammonium sulfate solution. The resulting coarse liposomes were sonicated with a probe sonicator for 5 min and were extruded through a series of polycarbonate membranes with pore sizes of 0.45 μm and 200 nm (five cycles for each). The external ammonium sulfate outside of the liposomes was removed by dialysis (MWCO 10 kDa) against a 10% glucose solution. DOX was actively loaded into the above liposomes using an ammonium sulfate gradient48 by incubating the liposomes with a DOX solution at 55 °C for 1 h. The unloaded DOX was removed by dialysis. Finally, various functionalized liposomes (L), ALN-HA-SS-L-L, HA-SS-L-L and HA-L-L, were prepared with a post-insertion method48 by incubating the above DOX-loaded liposomes at 55 °C for 30 min with ALN-HA-SS-L, HA-SS-L, and HA-L, respectively (polymer to total lipids ratio of 1.5: 98.5, mol/mol).


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In addition, PEGylated liposomes (PEG-L/DOX), a reference formulation simulating the clinically used Doxil, were prepared with the same process by post-insertion of DSPE-mPEG2000 (DSPE-mPEG2000/total lipid molar ratio 5:95). DiR-labeled liposomes were prepared by dissolving DiR in the organic solvents during thin film formation and using PBS for hydration. 2.4. Characterization of liposomal formulations 2.4.1. Particle size, zeta potential and morphology The particle size and zeta potential of liposomes in distilled water were determined by the dynamic light scattering (DLS) technique using a Malvern Zetasizer Nano ZS (Malvern, UK). The morphology of the liposomes was observed with transmission electron microscopy (TEM, JEM-1400, Japan).8 Briefly, a drop of liposome dispersion was placed on a 200-mesh copper grid and incubated for 2 min at room temperature. Then, the grid was stained with phosphotungstic acid solution, and images were taken after drying. 2.4.2. Encapsulation efficiency (EE) and drug loading (DL) To determine the EE and DL of DOX, the liposomes were disrupted with 10% Triton X-100. The amount of DOX in the liposomes was measured by fluorescence spectrophotometry (LS-55, Perkin Elmer, USA, excitation 488 nm, emission 593 nm). EE was calculated as the percentage of drug loaded in liposomes versus the drug amount used for loading, whereas DL was the percentage of drug in the drug-loaded liposomes. 2.5. Redox-responsiveness of liposomes The redox-sensitivity of ALN-HA-SS-L-L was assessed by monitoring the changes in size and morphology under reducing conditions8 using HA-L-L as a control. Briefly, liposomes were


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suspended in PBS (10 mM, pH 7.4) containing 10 mM dithiothreitol (DTT) followed by shaking at 100 rpm at 37 °C. The size and morphology were monitored at 1 and 3 h using DLS and TEM. To simulate cancer cell redox-induced drug release,8 1 ml of liposome suspension or DOX solution in dialysis bags (MWCO 10 kDa) was immersed in 30 ml of PBS (10 mM, pH 7.4) containing 20 μM or 10 mM GSH. The samples were kept under shaking at 37 °C, and the concentration of DOX in the release medium was monitored over 72 h. 2.6. In vitro cytotoxicity Human OS MG-63 cells were cultured in MEM containing 10% FBS, 1% antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin), 1% MEM nonessential amino acid solution, and 1% sodium pyruvate (100 mM). The cells were maintained at 37 °C in a humidified incubator under an atmosphere of 5% carbon dioxide and 95% oxygen. To compare the cytotoxicity of various DOX-encapsulated liposomes, liposomes were dissolved or suspended in culture medium without FBS and with final DOX concentrations ranging from 0.0001 to 10 μg/ml. DOX solutions acted as references. MG-63 cells (104/well) were seeded in 96-well plates and cultured overnight, and the culture medium was replaced with 100 μL of medium containing the DOX formulations. Following 24 h of incubation, cell viability was measured by the MTT assay. Cells without formulation treatment were used as a negative control (100% viability). The cytotoxicity of blank liposomes was also evaluated using the MTT assay. 2.7. Cellular uptake Cellular uptake of the various liposomes by MG-63 cells was compared using fluorescence inverted microscopy and flow cytometry. 2.7.1. Fluorescent inverted microscopy


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MG-63 cells were seeded in 6-well plates at a density of 2×105 cells/well. After 24 h, various liposomes at 20 μg/ml DOX equivalents were added (DOX acted as a fluorescent probe).8 An additional group of cells were treated with ALN-HA-SS-L-L/DOX as well as 0.5 mM free iRGD. After incubation for 30 min, 1 h, or 2 h, the cells were washed thrice with cold PBS and fixed with 4% paraformaldehyde solution for 0.5 h, and the nuclei were stained with Hoechst 33342 (10 mg/ml) for 10 min. After washing, the cells were observed using a fluorescence inverted microscope (Zeiss, Germany) with bright-field images indicating cell morphology. 2.7.2. Flow cytometry To further quantify the cellular uptake of free DOX or DOX-loaded liposomes, cells were treated with each of the formulations for 2 or 4 h. Then, the cells were centrifuged at 1500 rpm for 5 min and washed with cold PBS three times. The cells were resuspended in PBS and analyzed using an EPICS XL flow cytometer (Beckman, USA). 2.8. In vivo studies in orthotopic osteosarcoma mouse models 2.8.1. Establishment of an orthotopic human OS mouse model Orthotopic OS mouse models were established using a reported method.49,50 MG-63 cells at a confluence of 80% were detached and washed twice with PBS before being resuspended in PBS. Under sterile conditions, five-week-old female BALB/c nude mice were anesthetized with 4% chloral hydrate (0.1 ml/10 g). After decontamination, the cortical layer of the right tibia was perpendicularly pierced using a 22-gauge needle, followed by insertion of the needle approximately 3-5 mm into the diaphyseal shaft of the tibia. After removal of the needle, 5×106 MG-63 cells suspended in 50 μL of PBS were slowly injected in contact with the bone marrow with another 22-gauge needle. Upon completion of injection, gentle pressure was applied to the injection site to prevent cells from oozing out. Tibial tumor growth was observed every second


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day, and the tumor volume (V) was measured using a micrometer and calculated as 0.5 ab2, where ‘a’ and ‘b’ denote the longest and shortest diameters of the tumor, respectively. To confirm the OS model construction, eight animals were trialed with micro-computed tomographic (micro-CT) imaging. Two weeks post-tumor inoculation, mice were sacrificed, and their right tibiae were collected and placed in 10% neutral buffered formalin for 48 h before being stored in 70% ethanol until micro-CT scanning. The tibiae were assessed via radiographic analysis with a micro-CT scanner (NEMO micro-CT, PINGSENG Healthcare, China). CT images were taken at a resolution of 35 μm (achieved using 65 kV and 190 μA) and three dimensionally (3D) reconstructed with computer software (Recon; PINGSENG, Shanghai, China). 2.8.2. Efficacy study On day 15 post-inoculation, 42 tumor-bearing female nude mice showing tumors (100-150 mm3) were randomly divided into seven groups (n=6) and treated through tail vein injection of saline, free DOX, PEG-L/DOX, HA-L-L/DOX, HA-SS-L-L/DOX, or ALN-HA-SS-L-L/DOX (with or without 4 μmol/kg iRGD). The dose of DOX was 5 mg/kg. The animals were treated at 3 day intervals 3 times. Tumor volume, animal body weight and survival rate were monitored every 2 days. Animal survival curves were plotted using the Kaplan-Meier method, and the increase in life span (ILS) was calculated using the following equation: ILS (%) = (T/C-1) × 100 where T and C represent the mean survival time (in days) of the treated group and the control group, respectively.51 2.8.3. In vivo and ex vivo imaging To compare the bone/OS targeting properties of various liposomes, when tumors reached 250300 mm3 (18 days after inoculation), mice were randomly assigned to six groups (n=3). Each


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group was injected with free DiR, PEG-L/DiR, HA-L-L/DiR, HA-SS-L-L/DiR, or ALN-HA-SSL-L/DiR with or without iRGD at a DiR dose of 1 mg/kg. Fluorescence signals of DiR were captured under an in vivo imaging system (PerkinElmer IVIS Lumina, USA) with excitation at 745 nm and emission at 780 nm at 1-24 h post-administration. After the last imaging session, the mice were immediately sacrificed, and major organs (heart, liver, spleen, lung and kidney) and tumors were harvested for ex vivo imaging. 2.8.4. Histological evaluation To study the histopathology as a result of the treatments, tumor-bearing mice were randomly divided into 7 groups (n=5) and treated as described for the efficacy study. Three days after the last treatment, the mice were sacrificed. Tumors and major organs were harvested, fixed in 4% paraformaldehyde for 24 h at 4 °C and embedded in paraffin before being sliced. The sections (5 μm thick) were stained with hematoxylin-eosin (H&E) dyes for histopathological evaluation.52 Signs of tumor necrosis, cardiotoxicity of free DOX, and lung metastases (typically associated with OS2) were carefully observed. 2.9. Statistical analysis All data are expressed as the means ± standard deviations (SD). One-way analysis of variance (ANOVA) was used to determine the difference between groups, and data were considered significant at p < 0.05. Statistical comparisons of animal survival rate were performed using the log-rank test with GraphPad Prism 7 (online free trial version). 3. RESULT AND DISCUSSION Liposomes for step-by-step OS-targeted drug delivery were developed by surface coating with a new bone- and CD44-targeting redox-sensitive polymer, ALN-HA-SS-L. The dual-targeting ability of the liposomes was investigated in an OS cell model and orthotopic OS tumor animal


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models and compared with that of various reference liposomes, namely, liposomes with only HA or redox sensitivity and PEGylated liposomes. 3.1. Characterization of synthetic polymers Both the intermediate and final products were confirmed by proton nuclear magnetic resonance (1H-NMR) spectroscopy (Figure 2A-I).

Figure 2. 1H-NMR spectra of HA (A), ALN (B), HA-ALN (C), ALN-HA-CYS (D), HA-CYS (E), HA-HMDA (F), ALN-HA-SS-L (G), HA-SS-L (H), and HA-L (I). 400 MHz NMR, solvent: D2O.

3.1.1. Synthesis of CYS-HA-ALN, CYS-HA and HMDA-HA


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The successful synthesis of HA-ALN was confirmed in the 1H-NMR spectra of ALN and HAALN. Two characteristic peaks of ALN (Figure 2B), ‘a’ and ‘b,’ appeared at 1.92 ppm and 2.95 ppm, respectively. The peaks at 1.9 ppm and 3.12-4.51 ppm (Figure 2A) were due to the methyl protons of the acetylamide group and rings in HA, respectively. A new peak corresponding to the amide bond in the synthesized HA-ALN appeared at 2.70 ppm (Figure 2C), confirming the covalent conjugation of ALN to HA. Figure 2D and E show the characteristic peaks of ALN at ‘b’ (2.74 ppm) and CYS at ‘d’ (2.88 ppm, -CH2-SS-CH2-), confirming the chemical structures of CYS-HA-ALN and CYS-HA, respectively. The degree of substitution (DS) of the COOH groups of HA was found to be 9%, as calculated from the relative integrated intensities of the amide bond peak (2.74 ppm) and methyl protons of HA (peaks at ∼4.21-4.42 ppm)53 (Figure 2D). In other words, approximately 90% of the HA COOH groups in CYS-HA-ALN remained unsubstituted. The covalent conjugation of HMDA to HA was evidenced by the distinctive peak of HMDA present at ‘d’ (2.86 ppm) in the spectrum of HMDA-HA (Figure 2F). HMDA has a chain length similar to that of CYS but lacks the -SS- group, which makes HMDA-HA a perfect non-redoxsensitive reference polymer to CYS-HA. 3.1.2. Synthesis of the final products ALN-HA-SS-L, HA-SS-L and HA-L The formation of ALN-HA-SS-L, HA-SS-L and HA-L was confirmed by the 1H-NMR spectra (Figure 2G-L). The characteristic peaks of HA appeared at 1.91 ppm and 4.25-4.55 ppm, whereas the peaks corresponding to CYS and HMDA were found at ‘d’ (2.89 ppm) and ‘e’ (3.05 ppm), respectively. In addition, the peak of the PEG chain at 3.6 ppm was found in each of the final products. This result proves the successful synthesis of the amphiphilic multifunctional materials.


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3.2. Physicochemical characteristics of liposomes All types of liposomes displayed an average size of 150-170 nm with a relatively narrow distribution (PDI HA-L-L/DOX > PEG-L/DOX (Figure 5A). HA-L-L produced a smaller IC50 than PEG-L (1.75 vs 2.18 μg/ml), which is due to the use of HA. ALN-HA-SS-L-L/DOX and HA-SS-L-L/DOX produced similar IC50 values, which in turn were 1.3-1.5 times smaller than that of HA-L-L/DOX, confirming the beneficial effect of redox sensitivity. Interestingly, the IC50 value of ALN-HA-SS-L-L/DOX (1.35 μg/ml) was further reduced to 0.90 μg/ml by coincubation with iRGD.


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Figure 5. MG-63 cell viability of following exposure to (A) various DOX formulations and (B) blank liposomes (concentrations equivalent to DOX-loaded formulations) for 24 h. Consistent with other studies,8,9 free DOX induced a more prominent cytotoxic effect (IC50 0.63 μg/ml) than the liposomal formulations due to its immediate and rapid access to cells as a small molecule. However, the cell culture model would not reflect the in vivo tumor-targeting effect of the liposomes. Blank liposomes, including ALN-HA-SS-L-L/iRGD, showed little or no cytotoxicity against MG-63 cells (Figure 5B).


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3.5. Cellular uptake Following treatment of MG-63 cells with DOX formulations, HA-SS-L-L/DOX, or ALN-HASS-L-L/DOX, particularly ALN-HA-SS-L-L/DOX/iRGD, showed rapid cellular uptake (Figure 6A), and the fluorescence of DOX in cells was already saturated at 1 h, as there was no further increase when the treatment was extended to 2 h. Bright field images reveal some morphological change (round-shape) of the cells, possibly an early sign of cytotoxicity. Initially the uptake of HA-L-L/DOX and PEG-L/DOX was lower than that of the multifunctional liposomes, but at 2 h also reached the maximum level with little difference from other formulations. The fluorescence intensity of free DOX treated cells was initially low but reached the highest level at 1 h. The internalization of liposomes with functional groups, HA and -SS-, was quicker than free DOX at the early time point, owing to their high efficiency of drug transport via CD44-mediated endocytosis and intracellular release. However, it is well known that internalization of liposomes can be saturated over time. Additionally, consistent with the previous finding,8 bright field images show that the fluorescence of free DOX-treated cells was observed to be exclusively distributed near the nuclei, while liposomal DOX was also distributed in the cytoplasm, as the latter required an endosome-lysosome cellular trafficking process after internalization.


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Figure 6. Cellular uptake of DOX formulations. A) Fluorescent inverted microscope images and B) flow cytometry (n=3) of MG-63 cells exposed to various formulations for 0.5, 1, and 2 h, respectively (a: DOX solution, b: PEG-L/DOX, c: HA-L-L/DOX, d: HA-SS-L-L/DOX, e: ALNHA-SS-L-L/DOX, and f: ALN-HA-SS-L-L/DOX/iRGD).


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The lack of discrepancy in cellular uptake between HA-SS-L-L/DOX and ALN-HA-SS-LL/DOX was possibly attributable to the lack of hydroxyapatite on MG-63 cells. On the other hand, it is reported that the –COOH groups of HA play a central role in forming hydrogen bonds with the CD44 receptor55; these results indicate that the conjugate of ALN and PEG-lipid with HA, which occupied some of the –COOH groups of HA, did not negatively affect the CD44targeting property. The addition of iRGD further enhanced the rate and extent of cellular uptake of ALN-HA-SSL-L/DOX. One possible reason is that iRGD might be physically attached on the liposomal surface and might subsequently facilitate cellular uptake via integrin receptors, which are overexpressed on OS cells.41 Quantitative analysis of the cellular uptake by flow cytometry produced a similar but clearer trend as the fluorescent inverted microscopic observation with free DOX at 0.5 as an exception, (Figure 6B), possibly because flow cytometry picked up the fluorescence signal of DOX that was initially adsorbed on the cell membrane. Overall, these cellular uptake data appeared to be correlated with the cytotoxicity results. 3.6. In vivo studies 3.6.1. Confirmation of the establishment of an orthotopic OS model Micro-CT showed that seven of the eight examined animals had orthotopic OS growth at the shaft of the tibia around the inoculation site (Figure 7A). Notably, scientists are currently challenged with some ‘pitfalls’ in the clinical translation of nanomedicines, one of which is the use of subcutaneously implanted tumors.56,57 In this study, orthotopic OS models that may better reflect the physiopathology of OS in humans were constructed.


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Figure 7. Micro-CT image of a representative mouse leg collected two weeks after orthotopic injection of MG-63 cells (A) and the antitumor effect of different DOX formulations in OS tumorbearing mice (B-D): B) tumor growth curve, C) animal body weight, and D) Kaplan-Meier survival curves. **p < 0.01, ***p ALN-HA-SS-LL/DOX > HA-SS-L-L/DOX > HA-L-L/DOX > PEG-L/DOX > DOX, as shown by their ability to induce apoptosis of tumor cells.52 Free DOX treatment resulted in severe necrosis, breakage or loss of the myocardial fiber (typical signs of cardiotoxicity), and damage to the liver and kidney. These signs of toxicity were improved in the HA-L-L/DOX- and PEG-L/DOX-treated groups. In contrast, mice treated with ALN-HA-SS-L-L/DOX and ALN-HA-SS-L-L/DOX/iRGD displayed no pathological changes or inflammatory cell infiltration in the heart and kidney tissue sections. Minimal liver damage caused by ALN-HA-SS-L-L/DOX (but not when iRGD was coadministered) was noted, which is possibly related to the large size of the liposomes inducing nonspecific liver uptake (Figure 8B). Pulmonary hemorrhage was observed in only three groups, with the degree ranked as DOX > PEG-L/DOX > HA-L-L/DOX. Congestive hemorrhage was evident in the spleen tissues for all liposomal groups but was not as severe as that in the DOX group. It is envisaged that these pathological changes in the liver and spleen could be minimized as the liposomal size decreases by reducing the nonspecific accumulation of liposomes in these organs.


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Figure 9. Histological images of tumors and major organs 3 days after the last treatment, showing that free DOX treatment resulted in severe cardiotoxicity and damage to the liver and kidney, while ALN-HA-SS-L-L/DOX treatment suppressed lung metastasis. Photomicrographs are representative of five mice per group. Furthermore, a certain degree of metastasis to the lungs was detected in all animals except those treated with ALN-HA-SS-L-L/DOX with and without iRGD. Lung metastasis is a major reason for OS patient death.2 Notably, iRGD by itself has been reported to have anti-metastatic activity in mice.60 Overall, the animal studies suggest that the multifunctional liposomes not only suppressed tumor growth and protected health tissues but also inhibited lung metastases more effectively than traditional PEGylated liposomes.


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4. CONCLUSIONS In this study, bone- and CD44-dual-targeting redox-sensitive liposomes were developed by conjugating alendronate (ALN) and hyaluronic acid (HA) as targeting moieties onto the liposome surface via a bioreducible liker (-SS-). The conjugation of ALN with HA did not seem to reduce the CD44 binding ability of HA but did facilitate the localization of the liposomes at the OS site. In a preclinical model, the liposomes demonstrated great ability for specific intracellular drug delivery to OS and reduced the mortality of the animals. Moreover, in line with other reports, coadministration of iRGD augmented the tumor penetration of liposomes and consequently the efficacy of the liposomes, thus offering a simple yet effective approach to facilitate OS-targeted delivery of nanomedicines. ASSOCIATED CONTENT AUTHOR INFORMATION *Corresponding Author Dr. Zimei Wu (MSc, PhD) Mailing Address: School of Pharmacy, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand Tel: 64 9 923 1709 Fax: 64 9 3677192 Email: [email protected]

Author Contributions ZW and SF designed the experiments, analyzed and interpreted the results, and prepared the manuscript. SF conducted the experiments with some assistance from JL. ZXW and CG assisted


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with the CT image analysis and overall study design. ZZ assisted with the NMR experiments and data interpretation. ZW, HW and KS supervised the studies. All authors reviewed the manuscript. Funding Sources This study was funded by the Taishan Scholars Program of Shandong Province, China (No. tshw20130959), carried out at the School of Pharmacy, Yantai University. Notes The authors declare that they have no conflicts of interest to disclose. ACKNOWLEDGMENT The authors are grateful for the technical support in animal studies from Yantai Raphael Biotechnology Co., Ltd., the assistance with histological analysis by Dr. Guohua Yu from Yantai Yuhuangding Hospital and the valuable advice on chemical synthesis from Professor Jingtian Han (Binzhou Medical University, Yantai).


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REFERENCES (1)

Nedelcu, D.; Andreescu, N.; Boeriu, E.; Stefanescu, R.; Arghirescu, S.; Puiu, M.

Retrospective Study on Osteosarcoma and Ewing Sarcoma - our Experience. Maedica 2014, 9, 151–156. (2)

Ando, K.; Heymann, M. F.; Stresing, V.; Mori, K.; Redini, F.; Heymann, D. Current

Therapeutic Strategies and Novel Approaches in Osteosarcoma. Cancers 2013, 5, 591–616. (3)

Haghiralsadat, F.; Amoabediny, G.; Naderinezhad, S.; Zandieh-Doulabi, B.; Forouzanfar,

T.; Helder, M. N. Codelivery of Doxorubicin and JIP1 siRNA with Novel EphA2-targeted PEGylated Cationic Nanoliposomes to Overcome Osteosarcoma Multidrug Resistance. Int. J. Nanomed. 2018, 13, 3853–3866. (4)

Cui, C.; Yu, P.; Wu, M.; Zhang, Y.; Liu, L.; Wu, B.; Wang, C. X.; Zhuo, R. X.; Huang,

S. W. Reduction-sensitive Micelles with Sheddable PEG Shells Self-assembled from a Y-Shaped Amphiphilic Polymer for Intracellular Doxorubicine Release. Colloids Surf., B 2015, 129, 137– 145. (5)

Allen, T. M. Liposomal Drug Formulations Rationale for Development and What We

Can Expect for the Future. Drugs 1998, 56, 747–756. (6)

Huwyler, J.; Drewe, J.; Krähenbühl, S. Tumor Targeting Using Liposomal Antineoplastic

Drugs. Int. J. Nanomed. 2008, 3, 21–29. (7)

Wei, M.; Xu, Y.; Zou, Q.; Tu, L.; Tang, C.; Xu, T.; Deng, L.; Wu, C. Hepatocellular

Carcinoma Targeting Effect of PEGylated Liposomes Modified with Lactoferrin. Eur J Pharm Sci 2012, 46, 131–141.


33


(8)

Page 34 of 42

Chi, Y.; Yin, X.; Sun, K.; Feng, S.; Liu, J.; Chen, D.; Guo, C.; Wu, Z. Redox-sensitive

and Hyaluronic Acid Functionalized Liposomes for Cytoplasmic Drug Delivery to Osteosarcoma in Animal Models. J. Controlled Release 2017, 261, 113–125. (9)

Yin, X.; Chi, Y.; Guo, C.; Feng, S.; Liu, J.; Sun, K.; Wu, Z. Chitooligosaccharides

Modified Reduction-sensitive Liposomes: Enhanced Cytoplasmic Drug Delivery and Osteosarcomas-Tumor Inhibition in Animal Models. Pharm. Res. 2017, 34, 2172–2184. (10) Yin, X.; Feng, S.; Chi, Y.; Liu, J.; Sun, K.; Guo, C.; Wu, Z. Estrogen-functionalized Liposomes Grafted with Glutathione-responsive Sheddable Chotooligosaccharides for the Therapy of Osteosarcoma. Drug Delivery 2018, 25, 900–908. (11) Gilbert, H. F. Thiol/Disulfide Exchange Equilibria and Disulfde Bond Stability. Methods Enzymol. 1995, 251, 8–28. (12) Raina, S.; Missiakas, D. Making and Breaking Disulfide Bonds. Annu. Rev. Microbiol. 1997, 51, 179–202. (13) Balendiran, G. K.; Dabur, R.; Fraser, D. The Role of Glutathione in Cancer. Cell Biochem. Funct. 2004, 22, 343–352. (14) Schafer, F. Q.; Buettner, G. R. Redox Environment of the Cell as Viewed Through the Redox State of the Glutathione Disulfide/Glutathione Couple. Free Radical Biol. Med. 2001, 30, 1191–1212. (15) Wu, G.; Fang, Y.; Yang, S.; Lupton, J. R.; Turner, N. D. Glutathione Metabolism and its Implications for Health. J. Nutr. 2004, 134, 489–492.


34

Page 35 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(16) Kuppusamy, P.; Li, H.; Ilangovan, G.; Cardounel, A. J.; Zweier, J. L.; Yamada, K.; Krishna, M. C.; Mitchell, J. B. Noninvasive Imaging of Tumor Redox Status and its Modification by Tissue Glutathione Levels. Cancer Res. 2002, 62, 307–312. (17) Saito, G.; Swanson, J. A.; Lee, K. D. Drug Delivery Strategy Utilizing Conjugation via Reversible Disulfide Linkages: Role and Site of Cellular Reducing Activities. Adv. Drug Delivery Rev. 2003, 55, 199–215. (18) Barille, S.; Collette, M.; Bataille, R.; Amiot, M. Myeloma Cells Upregulate Interleukin-6 Secretion in Osteoblastic Cells Through Cell-to-Cell Contact but Downregulate Osteocalci. Blood 1995, 86, 3151–3159. (19) Magan-Fernandez, A.; Fernandez-Barbero, J. E.; O' Valle, F.; Ortiz, R.; Galindo-Moreno, P.; Mesa, F. Simvastatin Exerts Antiproliferative and Differentiating Effects on MG63 Osteoblast-like Cells: Morphological and Immunocytochemical Study. J. Periodontal Res. 2018, 53, 91–97. (20) Eliaz, R. E.; Nir, S.; Marty, C.; Szoka, F. C., Jr. Determination and Modeling of Kinetics of Cancer Cell Killing by Doxorubicin and Doxorubicin Encapsulated in Targeted Liposomes. Cancer Res. 2004, 64, 711–718. (21) Pouyani, T.; Prestwich, G. D. Functionalized Derivatives of Hyaluronic acid Oligosaccharides: Drug Carriers and Novel Biomaterials. Bioconjugate Chem. 1994, 5, 339–347. (22) Prestwich, G. D.; Marecak, D. M.; Marecek, J. F.; Vercruysse, K. P.; Ziebell, M. R. Controlled Chemical Modification of Hyaluronic acid: Synthesis, Applications, and Biodegradation of Hydrazide Derivatives. J. Controlled Release 1998, 53, 93–103.


35


Page 36 of 42

(23) Platt, V. M.; Szoka, F. C., Jr. Anticancer Therapeutics: Targeting Macromolecules and Nanocarriers to Hyaluronan or CD44, a Hyaluronan Receptor. Mol. Pharmaceutics 2008, 5, 474–486. (24) Maeda, H. Macromolecular Therapeutics in Cancer Treatment: the EPR Effect and Beyond. J. Controlled Release 2012, 164, 138–144. (25) Jiang, Y.; Wang, X.; Cheng, Y.; Peng, J.; Xiao, J.; Yi, Y. Associations between Inflammatory Gene Polymorphisms (TNF-α 308G/A, TNF-α 238G/A, TNF-β 252A/G, TGF-β1 29T/C, IL-6 174G/C and IL-10 1082A/G) and Susceptibility to Osteosarcoma: A Meta-Analysis and Literature Review. Oncotarget 2017, 8, 97571–97583. (26) Low, S. A.; Kopecek, J. Targeting Polymer Therapeutics to Bone. Adv. Drug Delivery Rev. 2012, 64, 1189–1204. (27) Sato, M.; Grasser, W.; Endo, N.; Akins, R.; Simmons, H.; Thompson, D. D.; Golub, E.; Rodan, G. A. Bisphosphonate Action. Alendronate Localization in Rat Bone and Effects on Osteoclast Ultrastructure. J. Clin. Invest. 1991, 88, 2095–2105. (28) Farrell, K. B.; Karpeisky, A.; Thamm, D. H.; Zinnen, S. Bisphosphonate Conjugation for Bone Specific Drug Targeting. Bone Reports 2018, 9, 47–60. (29) Liu, Y.; Jia, Z.; Akhter, M. P.; Gao, X.; Wang, X.; Wang, X.; Zhao, G.; Wei, X.; Zhou, Y.; Wang, X.; Hartman, C. W.; Fehringer, E. V.; Cui, L.; Wang, D. Bone-targeting Liposome Formulation of Salvianic acid A Accelerates the Healing of Delayed Fracture Union in Mice. Nanomedicine 2018, 14, 2271–2282.


36

Page 37 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(30) Wang, Y.; Huang, Q.; He, X.; Chen, H.; Zou, Y.; Li, Y.; Lin, K.; Cai, X.; Xiao, J.; Zhang, Q.; Cheng, Y. Multifunctional Melanin-like Nanoparticles for Bone-targeted Chemophotothermal Therapy of Malignant Bone Tumors and Osteolysis. Biomaterials 2018, 183, 10– 19. (31) Blattmann, C.; Thiemann, M.; Stenzinger, A.; Roth, E. K.; Dittmar, A.; Witt, H.; Lehner, B.; Renker, E.; Jugold, M.; Eichwald, V.; Weichert, W.; Huber, P. E.; Kulozik, A. E. Establishment of a Patient-derived Orthotopic Osteosarcoma Mouse Model. J. Transl. Med. 2015, 13, 136. (32) Killion, J. J.; Radinsky, P.; Fidler, I. J. Orthotopic Models are Necessary to Predict Therapy of Transplantable Tumors in Mice. Cancer Metastasis Rev. 1999, 17, 279–284. (33) Poh, S.; Chelvam, V.; Low, P. S. Comparison of Nanoparticle Penetration into Solid Tumors and Sites of Inflammation: Studies using Targeted and Nontargeted Liposomes. Nanomedicine 2015, 10, 1439–1449. (34) Sugahara, K. N.; Teesalu, T.; Karmali, P. P.; Kotamraju, V. R.; Agemy, L.; Girard, O. M.; Hanahan, D.; Mattrey, R. F.; Ruoslahti, E. Tissue-penetrating Delivery of Compounds and Nanoparticles into Tumors. Cancer Cell 2009, 16, 510–520. (35) Yin, H.; Yang, J.; Zhang, Q.; Yang, J.; Wang, H.; Xu, J.; Zheng, J. iRGD as a Tumorpenetrating Peptide for Cancer Therapy. Mol. Med. Rep. 2017, 15, 2925–2930. (36) Desgrosellier, J. S.; Cheresh, D. A. Integrins in Cancer: Biological Implications and Therapeutic Opportunities. Nat. Rev. Cancer 2010, 10, 9–22. (37) Ruoslahti, E. Specialization of Tumour Vasculature. Nat. Rev. Cancer 2002, 2, 83–90.


37


Page 38 of 42

(38) Clover, J.; Dodds, R. A.; Gowen, M. Integrin Subunit Expression by Human Osteoblasts and Osteoclasts in situ and in Culture. J. Cell Sci. 1992, 103, 267–271. (39) Gronthos, S.; Stewart, K.; Graves, S. E.; Hay, S.; Simmons, P. J. Integrin Expression and Function on Human Osteoblast-like Cells. J. Bone Miner. Res. 1997, 12, 1189–1197. (40) Avnet, S.; Longhi, A.; Salerno, M.; Halleen, J. M.; Perut, F.; Granchi, D.; Ferrari, S.; Bertoni, F.; Giunti, A.; Baldini, N. Increased Osteoclast Activity is Associated with Aggressiveness of Osteosarcoma. Int. J. Oncol. 1992, 33, 1231–1238. (41) Zhu, H.; Cai, H.; Tang, M.; Tang, J. Neuropilin-1 is Overexpressed in Osteosarcoma and Contributes to Tumor Progression and Poor Prognosis. Clin. Transl. Oncol. 2014, 16, 732–738. (42) Yu, H.; Tang, Z.; Song, W.; Zhang, D.; Zhang, Y.; Chen, X. Co-administration of iRGD Enhancing the Anticancer Efficacy of Cisplatin-loaded Polypeptide Nanoparticles. J. Controlled Release 2015, 213, e145–e146. (43) Morton, S. W.; Shah, N. J.; Quadir, M. A.; Deng, Z. J.; Poon, Z.; Hammond, P. T. Osteotropic Therapy via Targeted Layer-by-Layer Nanoparticles. Adv Healthc Mater 2014, 3, 867-875. (44) Xie, B.; Liu, A.; Fang, X.; Chen, Y.; Zhong, H. Rapid Determination of Alendronate to Quality Evaluation of Tablets by High Resolution (1)H NMR Spectroscopy. J. Pharm. Biomed. Anal. 2014, 93, 73–76. (45) Lee, H.; Mok, H.; Lee, S.; Oh, Y. K.; Park, T. G. Target-specific Intracellular Delivery of siRNA using Degradable Hyaluronic acid Nanogels. J. Controlled Release 2007, 119, 245–252.


38

Page 39 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(46) Yeom, J.; Chang, S.; Park, J. K.; Je, J. H.; Yang, D. J.; Choi, S. K.; Shin, H. I.; Lee, S. J.; Shim, J. H.; Cho, D. W.; Hahn, S. K. Synchrotron X-ray Bioimaging of Bone Regeneration by Artificial Bone Substitute of MegaGen Synthetic Bone and Hyaluronate Hydrogels. Tissue Eng., Part C 2010, 16, 1059–1068. (47) Hwang, T.; Han, H. D.; Song, C. K.; Seong, H.; Kim, J. H.; Chen, X.; Shin, B. C. Anticancer Drug-phospholipid Conjugate for Enhancement of Intracellular Drug Delivery. Macromol. Symp. 2007, 249-250, 109–115. (48) Zhang, W.; Wang, G.; See, E.; Shaw, J. P.; Baguley, B. C.; Liu, J.; Amirapu, S.; Wu, Z. Post-insertion of Poloxamer 188 Strengthened Liposomal Membrane and Reduced Drug Irritancy and in vivo Precipitation, Superior to PEGylation. J. Controlled Release 2015, 203, 161–169. (49) Curtis, R. C.; Custis, J. T.; Ehrhart, N. P.; Ehrhart, E. J.; Condon, K. W.; Gookin, S. E.; Donahue, S. W. Combination Therapy with Zoledronic acid and Parathyroid Hormone Improves Bone Architecture and Strength following a Clinically-Relevant Dose of Stereotactic Radiation Therapy for the Local Treatment of Canine Osteosarcoma in Athymic Rats. PLoS One 2016, 11, e0158005. (50) Uluckan, O.; Segaliny, A.; Botter, S.; Santiago, J. M.; Mutsaers, A. J. Preclinical Mouse Models of Osteosarcoma. BoneKEy Rep. 2015, 4, 670. (51) Wang, K.; Zhang, X.; Liu, Y.; Liu, C.; Jiang, B.; Jiang, Y. Tumor Penetrability and Antiangiogenesis using iRGD-mediated Delivery of Doxorubicin-polymer Conjugates. Biomaterials 2014, 35, 8735–8747.


39


Page 40 of 42

(52) Ma, S.; Zhou, J.; Zhang, Y.; He, Y.; Jiang, Q.; Yue, D.; Xu, X.; Gu, Z. Highly Stable Fluorinated Nanocarriers with iRGD for Overcoming the Stability Dilemma and Enhancing Tumor Penetration in an Orthotopic Breast Cancer. ACS Appl. Mater. Interfaces 2016, 8, 28468– 28479. (53) Zhang, Y.; Hu, Y.; Luo, Z.; Shen, X.; Mu, C.; Cai, K. Simultaneous Delivery of BMP-2 Factor and Anti-osteoporotic Drugs using Hyaluronan-assembled Nanocomposite for Synergistic Regulation on the Behaviors of Osteoblasts and Osteoclasts in vitro. J. Biomater. Sci., Polym. Ed. 2015, 26, 290–310. (54) Qhattal, H. S.; Hye, T.; Alali, A.; Liu, X. Hyaluronan Polymer Length, Grafting Density, and Surface Poly(Ethylene glycol) Coating Influence in vivo Circulation and Tumor Targeting of Hyaluronan-grafted Liposomes. ACS Nano 2014, 8, 5423–5440. (55) Nguyen, T. T.; Tran, D. P.; Pham Dinh Quoc, H.; Hoang, Z.; Carloni, P.; Van Pham, P.; Nguyen, C.; Li, M. S. Ligand Binding to Anti-Cancer Target CD44 Investigated by Molecular Simulations. J. Mol. Model. 2016, 22, 165. (56) Mitragotri, S.; Lammers, T.; Bae, Y. H.; Schwendeman, S.; De Smedt, S.; Leroux, J. C.; Peer, D.; Kwon, I. C.; Harashima, H.; Kikuchi, A.; Oh, Y. K.; Torchilin, V.; Hennink, W.; Hanes, J.; Park, K. Drug Delivery Research for the Future: Expanding the Nano Horizons and Beyond. J. Controlled Release 2017, 246, 183–184. (57) Lammers, T.; Kiessling, F.; Ashford, M.; Hennink, W.; Crommelin, D.; Storm, G. Cancer Nanomedicine: is Targeting our Target? Nat. Rev. Mater. 2016, 1, pii: 16069.


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(58) Jacevic, V.; Dragojevic-Simic, V.; Tatomirovic, Z.; Dobric, S.; Bokonjic, D.; Kovacevic, A.; Nepovimova, E.; Valis, M.; Kuca, K. The Efficacy of Amifostine against Multiple-Dose Doxorubicin-Induced Toxicity in Rats. Int. J. Mol. Sci. 2018, 19, 2370. (59) Dai, W.; Fan, Y.; Zhang, H.; Wang, X.; Zhang, Q.; Wang, X. A Comprehensive Study of iRGD-modified Liposomes with Improved Chemotherapeutic Efficacy on B16 Melanoma. Drug Delivery 2015, 22, 10–20. (60) Sugahara, K. N.; Braun, G. B.; De Mendoza, T. H.; Kotamraju, V. R.; French, R. P.; Lowy, A. M.; Teesalu, T.; Ruoslahti, E. Tumor-penetrating iRGD Peptide Inhibits Metastasis. Mol. Cancer Ther. 2015, 14, 120–128.


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Engineering of Bone- and CD44-Dual-Targeting Redox-Sensitive

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