Bone Targeted Delivery of SDF-1 via Alendronate Functionalized

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Biological and Medical Applications of Materials and Interfaces

Bone Targeted Delivery of SDF-1 via Alendronate Functionalized Nanoparticles in Guiding Stem Cells Migration Qingchang Chen, Chuping Zheng, Yanqun Li, Shaoquan Bian, Haobo Pan, Xiaoli Zhao, and William Weijia Lu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08606 • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on June 26, 2018

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ACS Applied Materials & Interfaces

Bone Targeted Delivery of SDF-1 via Alendronate Functionalized Nanoparticles in Guiding Stem Cells Migration

Qingchang Chen1#, Chuping Zheng1,3#, Yanqun Li1#, Shaoquan Bian1, Haobo Pan1, Xiaoli Zhao1*, William W. Lu2.

1

Research Center for Human Tissues and Organs Degeneration, Institute of

Biomedicine and Biotechnology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, PR China 2

Department of Orthopaedic and Traumatology, The University of Hong Kong, 21 Sassoon Rd., Pokfulam, Hong Kong, 999077, PR China

3

School of Pharmaceutical Science, Guangzhou Medical University, Guangzhou, Guangdong, 511436, PR China.

Key words: bone targeting, gene delivery, stem cell homing, SDF-1, osteoporosis therapy

* Corresponding author Dr Xiaoli Zhao, PhD Research Center for Human Tissues and Organs Degeneration, Shenzhen Institute of Advanced Technology, Chinese Academy of Science, Shenzhen, China Tel: +86 755 86585222 Fax: +86 755 86585233 Email: [email protected] #, These authors contributed equally to this work.

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Abstract Stem cells are well-known for great capacity of tissue regeneration. This provides a promising source for cell-based therapies in treating various bone degenerative disorders. However, the major hurdles for their application in transplantation are the poor bone marrow homing and engraftment efficiencies. Stromal cell-derived factor 1 (SDF-1) has been identified as a major stem cells homing factor. With the aims of bone targeted SDF-1 delivery and regulating MSCs migration, alendronate modified liposomal nanoparticles (Aln-Lipo) carrying SDF-1 gene were developed in this study. Alendronate modification significantly increased the mineral binding affinity of liposomes, and facilitated the gene delivery to osteoblastic cells. Up-regulated SDF-1 expression in osteoblasts triggered MSCs migration. Systemic infusion of Aln-Lipo-SDF-1 with fluorescence labeling in mice showed the accumulation in osseous tissue by biophotonic imaging. Corresponding to the delivered SDF-1, the transplanted GFP+ MSCs were attracted to bone marrow and contributed to bone regeneration. By targeted SDF-1 delivery. This study may provide a useful technique in regulating stem cells migration.

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1. Introduction Osteoporosis is a worldwide health problem related to the aging of population.1 Imbalanced bone remodeling favoring bone resorption over bone formation results in bone mineral loss.2,3 Drugs with antiresorptive and anabolic effects on osteoclast and osteoblast have been applied, however, the restoration of bone mass and strength has not yet been achieved.4 The decreases in the activities and numbers of osteoblasts and mesenchymal stem cells (MSCs) with aging are the main reasons leading to the reduced osteogenesis and bone formation.5,6 Stem cells are well-known for the great capacity for tissue regeneration.7,8 Because of the osteogenic potential as well as ease of isolation and expansion, they have become promising source for cell-based therapy in treating various bone degenerative disorders.9,10 Systemic MSCs transplantation has already been applied clinically for the treatment of osteogenesis imperfecta. Although short-term improvements were seen in several cases, long-term benefits in promoting an osteogenic response were not observed thus far.11,12 Poor bone marrow homing and low engraftment efficiencies are the major hurdles for systemic infusion of MSCs. MSCs are generally unable to home to the targeted tissue unless genetically modified or infused under injury conditions.13,14 Efforts have been made in guiding MSCs homing. A peptide sequence (E7) was identified with high affinity to bone marrow-derived MSCs, and it was conjugated on polycaprolactone meshes for cartilage repairing.15 LLP2A-Ale designed with dual targeting groups could direct MSCs to bone surface to augment bone formation.16 CD44 was also reported as a homing molecule for MSCs in bone trafficking.17 However, the challenge for regulating MSCs migration is the lack of specific phenotypic marker in identifying 3



MSCs. Increasing evidences suggest that MSCs could home to damaged tissues in response to injury.18-20 It’s a natural healing response with complex multistep processes, and regulated by many chemotactic factors.21 Among them, stromal cell-derived factor 1 (SDF-1) was identified as a major factor for regulating stem cells homing.22 It has been found up-regulated at injury sites and served as a potent chemoattractant to recruit MSCs. The transiently expressed SDF-1 in ischaemic cardiomyopathy model showed the attraction of stem cells to injured myocardium.23 In ischemic heart failure model, over expression of SDF-1 in myocardium could recruit endogenous cardiac stem cells and promoted cardiac function.24,25 Its repairing effect has also been demonstrated in other tissues such as skin, lung, liver, brain and cartilage.26-30 More importantly, age related decline of stem cell number in bone marrow was found related to the increase of bone marrow fat as well as the decrease of plasma SDF-1 level.31 Genetically manipulated MSCs with CXCR4 (SDF-1 receptor) expression could recover bone mass in osteoporotic mice by systemic transplantation.32 Inspired by the effect of SDF-1 in guiding stem cells homing, bone targeted nanoparticles carrying SDF-1 gene was developed in this study and expected to enhance stem cells recruitment for bone regeneration. Targeted drug delivery provides the benefits in improving therapeutic effect and reducing side effect. Bone targeting could be realized by modifying liposome with alendronate (Aln-Lipo). It has high affinity for bone mineral and has been widely studied in bone targeted drug delivery.33-36 The targeting effect of Aln-Lipo was examined in this study by mineral binding and tissue biodistribution. The stem cell migration corresponding to the delivered SDF-1 was studied by cell migration assay and GFP+ MSCs transplantation. 4


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2. Results and Discussion 2.1. Preparation of Alendronate Functionalized Liposomes. Non-viral vectors mediated systemic gene delivery has many advantages, particularly with respect to safety.37 Lipid-based gene carriers are among the most widely used non-viral vectors, and have been approved by FDA for clinical trials.38,39 For construction of the bone targeted liposomes, alendronate (Aln) conjugated lipid was synthesized via conventional carbodiimide reaction (Figure 1A) and incorporated into liposomes (Figure

1B).

The

reaction

was

conducted

between

carboxyl

group

of

DSPE-PEG2000-COOH and amino group of alendronate (Aln). The chemical structure of synthesized DSPE-PEG-Aln was confirmed by 1H-NMR (Figure 1C). Alendronate showed a characteristic signal at 2.0 ppm corresponding to CH2 protons, and this could also be found in the spectrum of DSPE-PEG-Aln. Lipid showed the representative signals at 1.26 ppm and 3.74 ppm corresponding to CH2 of the long hydrocarbon chain and CH2O of PEG respectively,40 confirmed the formation of DSPE-PEG-Aln conjugate. Liposomes (Lipo) were prepared by thin-film hydration method with the components of DOTAP, DOPE, Chol and DSPE-PEG at a mol% ratio of 63:16:16:5.41 In this formulation, DOTAP is a cationic lipid for gene carrying, DOPE is a neutral lipid as “helper lipid” to enhance delivering efficiency and stability of nanoparticle, and PEG usually helps in minimizing nonspecific interaction to increase circulation time.37 Aln functionalized liposomes (Aln-Lipo) were prepared similarly by replacing the component of DSPE-PEG with DSPE-PEG-Aln.

2.2 Characterization of Aln Functionalized Liposomes.

The prepared

liposomes were characterized for the size, zeta potential, morphology, DNA 5



condensation ability and encapsulation efficiency. The data from dynamic light scattering detector showed that the average hydrodynamic diameter of Lipo was around 116 nm. Aln modification slightly increased the hydrodynamic diameter to about 123 nm, but it did not significantly influence the zeta potential (Figure 2A). As presented in TEM images, Aln-Lipos were spherical nanoparticles with the size corresponding to the hydrodynamic diameter (Figure 2B). The gel electrophoresis result showed that the band of cDNA was undetected in the N/P ratio of 1:1 (Figure 2C), indicating the complete DNA condensation. The encapsulation efficiency (EE%) of Lipo-Aln was above 66%, studied by ethidium bromide assay as after complete condensation (Figure 2D). Bone microenvironment is rich in hydroxyapatite (HAp). The bone targeting effect of Aln-Lipo was evaluated in vitro by its binding affinity to HAp.40 Fluorescent liposomes with NBD component were incubated with HAp, and the binding affinity was measured by fluorescence spectrophotometer. Liposomes (Lipo) showed the HAp binding affinity around 14%. With alendronate modification, the HAp binding affinity of Aln-Lipo was significantly increased to 80% (Figure 2E), confirming the possible bone targeting potential.

2.3. In vitro Transfection Efficiency and Cytotoxicity. The gene delivery abilities of liposomes were formulated by the reporter gene expression in COS-1 cells and MC3T3-E1 osteoblastic cells. The N/P ratios of Lipo and Aln-Lipo to DNA had been optimized as 4:1. The luciferase gene expression in COS-1 cells was showed on Figure 3A. Lipo and Aln-Lipo showed significantly higher transfection efficiency than LipofectamineTM 2000 (Lipo2000). Aln modification did not have influence on efficiency in these cells. This was further studied by GFP gene transfection and 6


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compared with MC3T3-E1 osteoblastic cells (Figure 3B). Clearly GFP expression could be observed in these cells. The transfection rate was around 50% for COS-1 and 20% for MC3T3-E1 cells respectively, quantified by flow cytometry. The difference in the rate between these cells was due to their different transfectable properties. It was worth noting that Aln modification increased the efficiency around 10% in MC3T3-E1 cells from 15.1% to 24.4%. Whereas, the increment in COS-1 cells was not so significant as in MC3T3-E1 osteoblastic cells. It has been reported that Aln conjugation on nanoparticles could increase their cellular accumulation in osteoblastic cells.40 Protein tyrosine phosphatases expressed on the surface of osteoblastic cells were conceived as one of the possible bisphosphonate binding receptor.42 The enhancement in cellular internalization plays a pivotal role in efficient gene transfection. This result showed that Aln modification could improve the gene delivering ability of liposome in osteoblastic cells. The biocompatibility of Aln-Lipo was investigated on MC3T3-E1 osteoblastic cells by CCK-8 assay (Figure 3C). With increased concentration to 50 μg mL-1, both Lipo and Aln-Lipo could maintain the cell viability above 95%, but there barely were cells alive at this concentration in Lipo2000 group. DOSPA contained in Lipo2000 is a multivalent cationic lipid, which brings the cytotoxicity at high concentration.43 DOTAP used in this study as liposome component was initially developed with the consideration of decreasing toxicity.44 The biodegradable ester bonds in its structure further enhanced the biocompatibility. In this part, by the transfection and cytotoxicity evaluation, alendronate modification increased the gene delivering ability of liposome in osteoblastic cells without bringing the cytotoxicity.

2.4. In vitro MSCs Migration. Stem cells provide a promising approach 7



for regenerative medicine. In the natural healing process of injury, the recruitment of MSCs is usually required for the subsequent reparation.18 The recruitment is initiated by various signaling molecules released from injured tissue such as cytokines and chemokines.45 Binding of chemokines to their receptors on cell surface induces cellular responses and results in the homing of MSCs to the injury sites. 46,47 As a chemokine, stromal cell-derived factor 1 (SDF-1) has been demonstrated with important role in stem cell homing. It was observed highly expressed in the site of bone fracture.48 The migration of human MSCs to the bone injury could be observed in a dose-dependent manner induced by SDF-1. Blocking SDF-1 axis resulted in the inhibition of MSCs migration and delayed bone regeneration.49 Therefore, in this study, bone targeted SDF-1 gene delivery was expected to enhance the migration of stem cells to bone and promote bone regeneration. Upregulation of SDF-1 in MC3T3-E1 osteoblastic cells was achieved by SDF-1 gene delivering. Aln-Lipo showed the best effect in improving SDF-1 expression in osteoblastic cells as determined by qRT-PCR for mRNA expression (Figure 4A) and Western blot for protein expression (Figure 4B). The migration activity of MSCs corresponding to the expressed SDF-1 from osteoblast was investigated in a transwell culture system (Figure 4C). MC3T3-E1 osteoblastic cells were planted in the lower chamber and induced for SDF-1 expression. C3H10T1/2 stem cells were growing in the upper compartment. The migrated MSCs were stained by crystal violet and quantified spectrophotometrically. The results showed that SDF-1 plasmid delivered by Aln-Lipo showed the best effect in attracting MSCs migration (Figure 4D,E), and this was consistent with SDF-1 expression level determined by the gene delivery ability.

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2.5. In vivo Bone Targeted SDF-1 Delivery and MSCs Homing.

The effects

of Aln-Lipo-SDF-1 (AL-SDF1) system in bone targeting and MSCs homing were further investigated in vivo. The targeting effect was investigated by the biodistribution of systemic infused of AL-SDF1. Mice were intravenously (iv) administered with fluorescent EMA labeled pCMV-SDF-1. These mice were divided into four groups: pSDF-1, Lipo2000-SDF-1, Lipo-SDF-1 and Aln-Lipo-SDF-1 (Figure 5Aa). Six hours after infusion, the major organs were collected and biophotonic imaged for EMA biodistribution. It could be found that the intensity of intraosseous fluorescence signal was strongest in the group of Aln-Lipo among all groups (Figure 5B), indicating the accumulation level of delivered SDF-1 in bone.50 Except liver, the fluorescence signal was barely detectable in other organs in all of the treatment groups. Currently, a series of bone targeted drug delivery systems have been designed to improve the drug’s accumulation in bone. Without targeting strategies, less than 1% of injected small molecule drugs could reach bone tissue due to the rapid degradation and renal clearance.51 Nanoparticles (NPs) could usually increase the solubility, stability, and circulation time of these drugs.52 Bone targeted groups could further enhance the accumulation of NPs in bone. Various oligopeptides have been developed for precisely targeting to osteoblasts,53,54 bone-formation surface,41 and bone-resorption surface55-57 for the aims of suppressing osteoclast activity, augmenting fracture healing and enhancing bone formation. Bisphosphonate including alendronate is a kind of well-known bone-seeking agent for the strong binding affinity to hydroxyapatite.40 It distributes to both bone-formation and bone-resorption surfaces mediated by a P-C-P bond.58 In addition, it could also provide antiresorptive action in preventing bone loss.59 These properties make alendronate more suitable in our study for bone targeted delivery of SDF-1. 9



To investigate whether the delivered SDF-1 could guide MSCs migration, we performed a cell transplantation study. GFP+ MSCs were intravenously injected into mice via lateral tail vein after SDF-1 gene administration for 24 h (Figure 5Aa). One day after the transplantation, the distribution of GFP+ MSCs in osseous tissue was investigated by tissue immunofluorescence staining and flow cytometry. Fluorescent images showed that there were more GFP+ MSCs in the femoral bone marrow in Aln-Lipo group as shown in Figure 5C. The quantified result obtained from flow cytometry further confirmed that Aln-Lipo significantly increased the number of GFP+ MSCs in bone marrow (Figure 5D). It was worth noting that the accumulated GFP+ MSCs could be found in certain areas around the vessels (Figure 5E), which suggested that they might transmigrate from endothelium towards the bone morrow. The effect of Aln-Lipo-SDF-1 (AL-SDF1) system on bone regeneration was studied by analyzing bone microstructure. With cell transplantation, mice were treated with AL-SDF1 once a week for a month, and saline was used as control. The bone microstructure was investigated by Micro-CT after one month treatment (Figure 5Ab). As shown in Figure 5F, the reconstructed three-dimensional images of femur and vertebrae showed increased bone mass in AL-SDF1 treatment group. Structure parameters of BMD and BV/TV indicated that AL-SDF1 has the effect in promoting bone formation (Figure 5G, Table 1). The regenerated bone structure may be induced by the participation of both transplanted cells and endogenous stem cells. Collectively, these data showed that bone targeted SDF-1 delivery could guide MSCs homing and enhance bone formation. MSCs homing has attracted extensive attention, but the underlying mechanisms are not fully elucidated. It is defined as the arrest of MSCs within the vasculature of a tissue followed by transmigration across the endothelium. This course was conceived 10


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in a similar way with the well-studied leukocytes and hematopoietic stem cell migration.21 Initiated by injury induced chemokine induction, cells homing is a coordinated multistep process, including rolling, endothelial adhesion and eventual transendothelial migration mediated by selectins and integrins.60,61 For MSCs homing, it is postulated that signaling molecules released from injured tissue stimulate the mobilization of MSCs from their storage niche into the circulation. Subsequently, adhesion molecules expressed on the endothelium mediated the rolling and adhesion of MSCs. These cells then transmigrate, proliferate and differentiate so as to directly participate in tissue repair or modulate the local environment by secreting cytokines.62,63 In this study, alendronate functionalized liposomes were used for bone targeted SDF-1 gene delivery. This resorted to the chelating interaction between bisphosphonates of alendronate and calcium ion in bone mineral.64 We speculated that the transfected osteoblast on bone surface could secrete SDF-1 protein into bone marrow and form the gradient (Figure 6). The bone marrow stem cells could response to SDF-1 gradient and home to the bone surface. Influenced by the microenvironment of bone surface, stem cells would differentiate into osteoblast and contribute to the bone regeneration. Bone targeted SDF-1 delivery provided a novel approach in improving MSCs homing to promote an osteogenic response utilizing either endogenous MSCs or systemic infused MSCs. This technique may help in stem cells-based therapy for treating various degenerative disorders clinically. Comparing with other reported methods in regulating stem cells homing, it has several advantages. The released SDF-1 could recruit stem cells by concentration gradient rather than direct contact as the affinity peptide functioned, which takes effect at larger area and attracts more stem cells.15,16 In addition, this system serves as a versatile platform. By using 11



different targeting moieties, this system could regulate MSCs homing to different organs. The success of this technique mainly depends on the efficiency of targeting delivery system and sufficient SDF-1 expression. It is subject to several barriers such as serum inactivation, enzymatic degradation, blood clearance, and reticuloendothelial system (RES) recognition in circulation after intravenous injection.65 These extracellular barriers are needed to overcome for increasing the amount of delivered SDF-1 to the targeted bone tissue to elicit a desired response. New techniques in prolonging circulation time, enhancing tissue targeting, and increasing gene delivery efficiency would help in optimizing this system to further improve the therapeutic effect. This study has demonstrated bone targeted SDF-1 delivery could direct MSCs homing and enhance bone formation on a normal mice model with MSCs transplantation. Its application on an osteoporosis model and the long-term effect are worth of further investigation.

3. Conclusion Alendronate functionalized liposome (Aln-Lipo) was developed in this study for bone targeted SDF-1 gene delivery and stimulating stem cells migration. Aln-Lipo complexed SDF-1 plasmid into nanoparticles around 120 nm. These nanoparticles showed strong binding affinity to hydroxyapatite, and facilitated the gene transfection in osteoblastic cells. Systemic delivering SDF-1 gene by Aln-Lipo showed increased accumulation in osseous tissue, followed by the attraction of transplanted MSCs participating in bone regeneration. The versatile drug loading property of Aln-Lipo provides an excellent platform for bone targeted delivery. Furthermore, SDF-1 loaded nanoparticle and subsequent targeted delivery may emerge as novel technique in guiding MSCs migration, which is not currently possible in MSCs transplantation. 12


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4. Experimental Section 4.1.

Materials.

1,2-dioleoyl-3-trimethylammonium-propane

(DOTAP),

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

(DOPE),

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]

(DSPE-PEG2000),

(DSPE-PEG-COOH)

and

DSPE-PEG2000

7-nitrobenz-2-oxa-1,3-diazol-4-yl

carboxylic

acid

phosphatidylcholine

(NBD-PC) were obtained from Avanti Polar Lipids. Cholesterol (Chol), alendronate (Aln), 2-(N-morpholino)ethanesulfonic acid (MES), 1-hydroxybenzotriazole (HOBt), N'-(3-dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride (EDC), deuterium oxide (D2O), and deuterochloroform (CDCl3) were obtained from Sigma-Aldrich. Lipofectamine™ 2000 was obtained from Invitrogen. Ethidium monoazide bromide (EMA) was obtained from Molecular Probes. Plasmids of pGL3-control (Promega) and pEGFP-N1 (Clontech) were used as reporter gene for luciferase and green fluorescent protein (GFP) expression. pCMV-SDF-1 was constructed in the lab encoding SDF-1 gene. These plasmids were expanded in Escherichia coli DH5α strain, purified by Pure Yield™ Plasmid Midiprep System (Promega) and stored in Tris-EDTA buffer at -20 °C. EMA labeled plasmid DNA was prepared by 1h UV exposure and series purification according to the manufacture’s instruction. 4.2. Cell Lines and Cell Culture. MC3T3-E1 murine calvarial osteoblasts were maintained in alpha-modified minimum essential medium (α-MEM, Hyclone) supplemented

with

10%

fetal

bovine

serum

(FBS,

Corning)

and

1%

penicillin-streptomycin (Hyclone) at 37 °C in a humidified atmosphere of 5% CO2. African green monkey kidney cells (COS-1) were cultured in Dulbecco’s 13



modified

Eagle

medium

(DMEM,

Hyclone),

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10%

FBS

and

1%

penicillin-streptomycin. Murine mesenchymal C3H10T1/2 cells and GFP+ bone marrow mesenchymal stem cell from C57BL/6 mouse (OriCell, Cyagen Bioscience) were cultured in Iscove’s modified Dulbecco’s medium (IMDM, Invitrogen) containing 10% FBS and 1% penicillin-streptomycin. Osteogenic medium was prepared by α-MEM withβ-glycerol phosphate (20 mM, Sigma), dexamethasone (1 nM, Sigma) and ascorbate 2-phosphate (0.5 μM, Sigma). The multi-differentiation potential of GFP+ stem cells was determined by osteogenic, adipogenic, and chondrogenic differentiation induced by different culture media (Figure S1). 4.3. Synthesis of the DSPE-PEG-Aln Conjugate. Alendronate (Aln) was conjugated to DSPE-PEG2000-COOH via carbodiimide chemistry using EDC as a coupling agent.66,67 Specifically, a solution of DSPE-PEG-COOH in MES buffer (50 mM, pH 5.5) was activated for 3 h at 0 °C by adequate amounts of EDC in the presence

of

HOBt

(DSPE-PEG2000-COOH:EDC:HOBt

=

0.03:1.25:2.1,

μmol:μmol).67 Aln (0.03 μmol) was then added to react with COOH group of DSPE-PEG2000-COOH (1.25 μmol) and incubated at room temperature under magnetic stirring for about 8 h (Fig.1A). The final product was purified by dialysis (MW cutoff of 1 kDa) for 3 days and lyophilized. The chemical structure of obtained DSPE-PEG-Aln

conjugate

was

confirmed

by

1

H-NMR

spectroscopy

(Bruker AVANCEIII, 400 MHz, CDCl3). 4.4. Preparation and Characterization of Aln-Functionalized Liposomes. Liposomes were prepared using the thin-film hydration method.41 DOTAP, DOPE, Chol and DSPE-PEG2000 were dissolved in chloroform at a mol% ratio of 63:16:16:5 and dried into a thin film by evaporating under negative pressure using a rotary evaporator at 60 °C.

33

The obtained lipid film was hydrated with 10 mM phosphate 14


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buffer saline (PBS, pH 7.4) pre-incubated in water bath at 60 °C to form multilamellar vesicles (MLVs). The resulted MLVs were then extruded in an Avanti mini extruder (Avanti Polar Lipids) 10 times through 0.2 μm polycarbonate membranes (Whatman) and 10 times through 0.1 μm membranes in stepwise manner to form larger unilamellar vesicles (LUV). Alendronate modified liposome (Aln-Lipo) was prepared similarly by replacing the component of DSPE-PEG2000 with DSPE-PEG-Aln. Fluorescent liposome was prepared by adding NBD-PC (0.2%, molar ratio) into the lipids during preparation. The liposomes with a final concentration of 1mg mL-1 were sterilized by passing through a 0.22 μm sterile filter and stored at 4 °C. Liposome-DNA complexes were prepared by mixing liposomes with DNA plasmids in a desired ratio and incubated at room temperature for 30 min. The hydrodynamic diameter and zeta potential of liposomes were measured by dynamic light scattering detector (Zetasizer Nano ZS, Malvern) following their dilution with distilled water. The morphology of liposomes was observed by transmission electron microscopy (TEM, Tecnai G2 F20 S-Twin). The DNA condensation ability of liposomes was investigated by gel retardation using agarose gel (1 %) containing ethidium bromide running at 80 V for 30 min in Tris-acetate-EDTA buffer. Each well contained DNA (0.5 μg) and liposomes at various N/P ratios. The encapsulation efficiency of DNA in liposomes was studied by ethidium bromide assay, calculated by the decrease of intensity protected by liposomes from ethidium staining.68 4.5. Hydroxyapatite Binding Affinity. The affinity of Aln functionalized liposomes to bone was investigated in vitro by hydroxyapatite (HAp) binding assay using fluorescent liposome containing NBD-PC (NBD-Lipo).41 Liposomes with and without alendronate modification were incubated with HAp (10 mg mL-1) 15



suspensions with gently shaken for 5 h at 25 °C. After centrifugation at 4000 rpm for 10 min to spin down HAp and liposomes bound to them, the fluorescence intensities of the supernatants were measured using fluorimetry (Edinburgh Instruments FS920, Ex 474 nm, Em 533 nm). The decrease in the intensity relative to the initial intensity suggests the degree of liposomes bound to HAp. 4.6. Transfection Efficiency and Cell Viability. The transfection activities of liposomes were investigated by delivering reporter gene of luciferase and GFP. COS-1 cells were firstly used to optimize and evaluate the luciferase expression. Cells were seeded in a 24-well plate at a density of 6 × 104 cells per well in complete medium for 24 h. Then cells were transfected with liposome/DNA complexes (1 μg DNA, 50 μL) in serum-free DMEM for 6 h, and further incubated in complete medium for 42 h. The transfection efficiency was determined by the relative luciferase activity (RLU) normalized by the protein content. The relative luciferase activity was evaluated by incubating cell lysate with luciferin substrate (Promega) and detected on luminometer (GloMax 96, Promega). The protein content was determined by Pierce™ BCA Protein Assay Kit (Thermo) on a Microplate reader (BioTek Synergy4) according to the manufacturer’s instructions. All the experiments were carried out in triplicate. GFP expression in COS-1 and MC3T3-E1 cells was used to further study the transfection efficiency by observing under inverted microscope (IX 71, Olympus) and quantified by flow cytometry (BD Ari III). MC3T3-E1 osteoblastic cells were cultured in osteogenic medium for osteogenic induction. The cell biocompatibility of liposomes was measured by the cell viability using cell counting kit-8 assay (CCK-8, Beyontime). In brief, MC3T3-E1 cells were cultured in 96-well plate with a concentration of 1 × 104 cells per well for 24 h, and then incubated with liposomes in different concentrations (2, 6, 10, 50 μg mL-1) for 48 16


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h. The plate was measured after 4 h incubation with CCK-8 solution on a Microplate reader at 450 nm. The cell viability was calculated as percentage of co-cultured cells subjected to untreated cells averaged from six independent experiments. 4.7. SDF-1 Delivery and MSCs Migration. After optimizing the transfection condition, pCMV-SDF1 plasmid was delivered to MC3T3-E1 osteoblastic cells. The expressed

SDF-1

mRNA

and

protein

were

measured

by

quantitative real-time RT-PCR (qRT-PCR) and Western blot. The total RNA was extracted with Trizol (Invitrogen) after 24 h transfection, and then reversely transcribed into cDNA using iScript cDNA Synthesis Kit (Thermo). The qRT-PCR was performed on CFX96 Real-Time System (Bio-rad), with the forward primer 5′-CGGGGTACCGCCACCATGGAAGACGCCAAAAACAT-3′ and the reverse primer 5′-CCGGAATTCCTAGAATTACACGGCGAT-3′. For examining SDF-1 protein expression, cells were harvested and lysed after 48 h transfection. The cell lysates with equal amount of proteins were separated by SDS-PAGE and blotted onto a nitrocellulose membrane (Bio-Rad Laboratories). The proteins were probed with anti-SDF-1 (Cell signaling) and anti-β-Actin (Cell signaling) antibodies, followed by a horseradish peroxidash (HRP)-conjugated secondary antibody (Earthox). The positive bands were visualized by enhanced chemiluminescence (ECL) solution (Amersham Biosciences) plus detection system. To investigate the migration of stem cells in response to the expressed SDF-1, a transwell assay was performed on a 24-transwell chambers (Corning). MC3T3-E1 cells were seeded in the lower chamber with a concentration of 1 × 104 and pre-incubated in osteogenic medium. Twenty-four hours after transfection of MC3T3-E1 cells with pCMV-SDF-1, mesenchymal C3H10T1/2 stem cells were planted in the upper chamber with a density of 2 × 104 cells per well and further 17



incubated for 24 h. Cells stayed on the upper surface of porous filter (8 μm) were removed with a cotton swab, and migrated cells to the lower surface were observed. These cells were stained with 0.5% crystal violet and observed under a light microscope. The results were quantified by dissolving crystal violet in 33% acetic acid and measured at 573 nm on a Microplate reader. 4.8. Bone-selective Delivery for MSCs Homing in vivo. The bone targeting effect of Aln-Lipo-SDF-1 (AL-SDF1) system was evaluated in vivo by observing the biodistribution of the delivered EMA labeled SDF-1 gene after systemic infusion (Fig. 5Aa). C57BL/6 mice (5-6 weeks) were obtained from Medical Laboratory Animal Center (China) and acclimatized for one week at animal housing facility. The animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Shenzhen Institutes of Advanced Technology, the Chinese Academy of Sciences. Mice were divided into four groups, and subjected to SDF-1 plasmid DNA, LipofectamineTM 2000-SDF-1 (Lipo2000), liposomes-SDF-1 (Lipo) and Aln modified liposomes-SDF-1 (Aln-Lipo) respectively. The complexes containing 20 μg EMA-pCMV-SDF-1 DNA were suspended in saline (125 μL) and administrated via lateral tail vein injection. Six hours later, the mice (n=3 in each group) were sacrificed and the major organs (heart, liver, spleen, lung, kidney and femur) were collected for EMA fluorescence detection using Xenogen IVIS imaging system (Caliper IVIS Spectrum, USA). The attraction effect of the delivered SDF-1 in stem cells homing to bone was investigated by GFP+ MSCs transplantation (Fig. 5Aa). GFP+ MSCs (5×105) in 10 L of IMDM were injected via lateral tail vein 24 hours after Aln-Lipo-SDF-1 administration.69 The mice were euthanized 24 h after cell transplantation, and the distal femora were collected for immunofluorescence staining to observe the GFP + 18


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MSCs distribution (n=3 in each group). The number of GFP+ cells in bone marrow was quantified by flow cytometry analysis (Becton Dickinson, n=5 in each group). 4.9. Immunofluorescence Histology. At the determined time, mice were euthanized, and the femora were resected. Specimens were fixed in PBS containing 4% paraformaldehyde for 2 days at 4 °C before decalcification with 10% ethylenediaminetetraacetic acid (EDTA) for 10 days. Then, the specimens were embedded with optimal cutting temperature compound (OCT, SAKURA) and cut into 5 μ sections. Immunofluorescent staining was performed on sections with anti-GFP antibody (abcam) followed by FITC conjugated secondary antibody (Vector Laboratories). The sections were mounted with the medium containing DAPI (Vector Laboratories) and observed under a fluorescence microscope (Olympus BX53). 4.10. Micro-CT Analysis. The effect of Aln-Lipo-SDF-1 (AL-SDF1) on bone regeneration was evaluated by investigating the bone microstructure via micro computed tomography (micro-CT) imaging analysis. Ten C57BL/6 mice (5-6 weeks) were divided into two groups with the treatment of saline (control) or AL-SDF1 once a week (Fig. 5Ab). After four weeks, the femora and lumbar vertebral bodies (Lv5) were harvested and fixed in 10% neutral buffered formalin (Fisher) for 24 h at room temperature. The bone microstructure of specimens were scanned on high-resolution micro-CT (SkyScan1176, Belgium) at a voltage of 60 kVp, a current of 417 μA, a resolution of 9.0 μm pixel-1. Software of NRecon, CTAn and μCTVol was used for three-dimensional reconstruction (threshold 80) and analyzing the parameters. Eighty continuous slices in the region of distal femur above the growth plate were chosen to investigate the microarchitecture. For the lumbar vertebral bodies, the entire region of Lv5 body was analyzed. The structure parameters of trabecular bone including bone mineral density (BMD), bone volume/tissue volume (BV/TV), Tb.Th (trabecular 19



thickness) and Tb.N (trabecular numbers) were calculated after three-dimension reconstruction 4.11. Statistical Analysis. Statistical analyses were performed by SPSS software. Data in the results are presented as mean ±standard deviation (S.D.). Statistical significance was evaluated using Student's t test or analysis of variance (ANOVA), and p < 0.05 was considered statistically significant.

ASSOCIATED CONTENT: Supporting Information. Characterization of multi-differentiation potential of GFP+ stem cells by Alizarin Red S staining, Oil Red O staining and alcian blue staining.

Acknowledgements This work was funded by National Natural Science Foundation of China (81672226), Science and Technology Research Funding of Shenzhen (JCYJ20160531174634936, JCYJ20170413162540673), Shenzhen Peacock Program (110811003586331) and Science and technology project of Guangdong (2016A020222007).

Conflict of Interest The authors declare no conflict of interest.

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Reference

(1) Adler, R. A.; El-Hajj Fuleihan, G.; Bauer, D. C.; Camacho, P. M.; Clarke, B. L.; Clines, G. A.; Compston, J. E.; Drake, M. T.; Edwards, B. J.; Favus, M. J.; Greenspan, S. L.; McKinney, R.; Pignolo, R. J.; Sellmeyer, D. E. Managing Osteoporosis in Patients on Long-Term Bisphosphonate Treatment: Report of a Task Force of the American Society for Bone and Mineral Research. J. Bone Miner. Res. 2016, 31, 16-35. (2) 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. (3) Appelman-Dijkstra, N. M.; Papapoulos, S. E. Modulating Bone Resorption and Bone Formation in Opposite Directions in the Treatment of Postmenopausal Osteoporosis. Drugs 2015, 75, 1049-1058. (4) Cosman, F.; Nieves, J. W.; Dempster, D. W. Treatment Sequence Matters: Anabolic and Antiresorptive Therapy for Osteoporosis. J. Bone Miner. Res. 2017, 32, 198-202. (5) Antebi, B.; Pelled, G.; Gazit, D. Stem Cell Therapy for Osteoporosis. Curr. Osteoporos. Rep. 2014, 12, 41-47. (6) Kiernan, J.; Hu, S.; Grynpas, M. D.; Davies, J. E.; Stanford, W. L. Systemic Mesenchymal Stromal Cell Transplantation Prevents Functional Bone Loss in A Mouse Model of Age-Related Osteoporosis. Stem Cells Transl. Med. 2016, 5, 683-693. (7) Murphy, M. B.; Moncivais, K.; Caplan, A. I. Mesenchymal Stem Cells: Environmentally Responsive Therapeutics for Regenerative Medicine. Exp. Mol. Med. 2013, 45, e54. (8) Bianco, P.; Cao, X.; Frenette, P. S.; Mao, J. J.; Robey, P. G.; Simmons, P. J.; Wang, C.-Y. The Meaning, the Sense and the Significance: Translating the Science of Mesenchymal Stem Cells into Medicine. Nat. Med. 2013, 19, 35-42. (9) Descalzo, D. L. M.; Ehnes, D. D.; zur Nieden, N. I. Stem Cells for Osteodegenerative Diseases: Current Studies and Future Outlook. Regen. Med. 2014, 9, 219-230. (10) Mobasheri, A.; Kalamegam, G.; Musumeci, G.; Batt, M. E. Chondrocyte and Mesenchymal Stem Cell-Based Therapies for Cartilage Repair in Osteoarthritis and Related Orthopaedic Conditions. Maturitas 2014, 78, 188-198. (11) Horwitz, E. M.; Prockop, D. J.; Fitzpatrick, L. A.; Koo, W. W. K.; Gordon, P. L.; Neel, M.; Sussman, M.; Orchard, P.; Marx, J. C.; Pyeritz, R. E.; Brenner, M. K. Transplantability and Therapeutic 21



Effects of Bone Marrow-derived Mesenchymal Cells in Children with Osteogenesis Imperfecta. Nat. Med. 1999, 5, 309-313. (12) Horwitz, E. M.; Gordon, P. L.; Koo, W. K. K.; Marx, J. C.; Neel, M. D.; McNall, R. Y.; Muul, L.; Hofmann, T. Isolated Allogeneic Bone Marrow-derived Mesenchymal Cells Engraft and Stimulate Growth in Children with Osteogenesis Imperfecta: Implications for Cell Therapy of Bone. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 8932-8937. (13) Gutwald, R.; Haberstroh, J.; Kuschnierz, J.; Kister, C.; Lysek, D. A.; Maglione, M.; Xavier, S. P.; Oshima, T.; Schmelzeisen, R.; Sauerbier, S. Mesenchymal Stem Cells and Inorganic Bovine Bone Mineral in Sinus Augmentation: Comparison with Augmentation by Autologous Bone in Adult Sheep. J. Oral. Max. Surg. 2010, 48, 285-290. (14) Granero-Molto, F.; Weis, J. A.; Miga, M. I.; Landis, B.; Myers, T. J.; O'Rear, L.; Longobardi, L.; Jansen, E. D.; Mortlock, D. P.; Spagnoli, A. Regenerative Effects of Transplanted Mesenchymal Stem Cells in Fracture Healing. Stem Cells 2009, 27, 1887-1898. (15) Shao, Z. X.; Zhang, X.; Pi, Y. B.; Wang, X. K.; Jia, Z. Q.; Zhu, J. X.; Dai, L. H.; Chen, W. Q.; Yin, L.; Chen, H. F.; Zhou, C. Y.; Ao, Y. F. Polycaprolactone Electrospun Mesh Conjugated with An MSC Affinity Peptide for MSC Homing In Vivo. Biomaterials 2012, 33, 3375-3387. (16) Guan, M.; Yao, W.; Liu, R.; Lam, K. S.; Nolta, J.; Jia, J.; Panganiban, B.; Meng, L.; Zhou, P.; Shahnazari, M.; Ritchie, R. O.; Lane, N. E. Directing Mesenchymal Stem Cells to Bone to Augment Bone Formation and Increase Bone mass. Nat. Med. 2012, 18, 456-462. (17) Sackstein, R.; Merzaban, J. S.; Cain, D. W.; Dagia, N. M.; Spencer, J. A.; Lin, C. P.; Wohlgemuth, R. Ex Vivo Glycan Engineering of CD44 Programs Human Multipotent Mesenchymal Stromal Cell Trafficking to Bone. Nat. Med. 2008, 14, 181-187. (18) Fong, E. L.; Chan, C. K.; Goodman, S. B. Stem Cell Homing in Musculoskeletal Injury. Biomaterials 2011, 32, 395-409. (19) Chen, Y.; Xiang, L. X.; Shao, J. Z.; Pan, R. L.; Wang, Y. X.; Dong, X. J.; Zhang, G. R. Recruitment of Endogenous Bone Marrow Mesenchymal Stem Cells Towards Injured Liver. J. Cell Mol. Med. 2010, 14, 1494-1508. (20) Chapel, A.; Bertho, J. M.; Bensidhoum, M.; Fouillard, L.; Young, R. G.; Frick, J.; Demarquay, C.; Cuvelier, F.; Mathieu, E.; Trompier, F.; Dudoignon, N.; Germain, C.; Mazurier, C.; Aigueperse, J.; Borneman, J.; Gorin, N. C.; Gourmelon, P.; Thierry, D. Mesenchymal Stem Cells Home to Injured 22


Page 22 of 39

Page 23 of 39 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


Tissues When Co-infused with Hematopoietic Cells to Treat A Radiation-induced Multi-organ Failure Syndrome. J.Gene Med. 2003, 5, 1028-1038. (21) Karp, J. M.; Leng Teo, G. S. Mesenchymal Stem Cell Homing: the Devil is in the Details. Cell Stem Cell 2009, 4, 206-216. (22) Ghadge, S. K.; Muhlstedt, S.; Ozcelik, C.; Bader, M. SDF-1 Alpha as A Therapeutic Stem Cell Homing Factor in Myocardial Infarction. Pharmacol. Therapeut. 2011, 129, 97-108. (23) Askari, A. T.; Unzek, S.; Popovic, Z. B.; Goldman, C. K.; Forudi, F.; Kiedrowski, M.; Rovner, A.; Ellis, S. G.; Thomas, J. D.; DiCorleto, P. E.; Topol, E. J.; Penn, M. S. Effect of Stromal-Cell-Derived Factor 1 on Stem-Cell Homing and Tissue Regeneration in Ischaemic Cardiomyopathy. Lancet 2003, 362, 697-703. (24) Unzek, S.; Zhang, M.; Mal, N.; Mills, W. R.; Laurita, K. R.; Penn, M. S. SDF-1 Recruits Cardiac Stem Cell-Like Cells that Depolarize In Vivo. Cell Transplant. 2007, 16, 879-886. (25) Sundararaman, S.; Miller, T. J.; Pastore, J. M.; Kiedrowski, M.; Aras, R.; Penn, M. S. Plasmid-Based Transient Human Stromal Cell-Derived Factor-1 Gene Transfer Improves Cardiac Function in Chronic Heart Failure. Gene Ther. 2011, 18, 867-873. (26) Zhu, Y.; Hoshi, R.; Chen, S.; Yi, J.; Duan, C.; Galiano, R. D.; Zhang, H. F.; Ameer, G. A. Sustained Release of Stromal Cell Derived Factor-1 From an Antioxidant Thermoresponsive Hydrogel Enhances Dermal Wound Healing in Diabetes. J. Control Release 2016, 238, 114-122. (27) Rafii, S.; Cao, Z.; Lis, R.; Siempos, II; Chavez, D.; Shido, K.; Rabbany, S. Y.; Ding, B. S. Platelet-Derived SDF-1 Primes the Pulmonary Capillary Vascular Niche to Drive Lung Alveolar Regeneration. Nat. Cell Biol. 2015, 17, 123-136. (28) Chen, P.; Tao, J.; Zhu, S.; Cai, Y.; Mao, Q.; Yu, D.; Dai, J.; Ouyang, H. Radially Oriented Collagen Scaffold with SDF-1 Promotes Osteochondral Repair by Facilitating Cell Homing. Biomaterials 2015, 39, 114-123. (29) Lei, Y.; Liu, Z.; Han, Q.; Kang, W.; Zhang, L.; Lou, S. G-CSF Enhanced SDF-1 Gradient Between Bone Marrow and Liver Associated with Mobilization of Peripheral Blood CD34+ Cells in Rats with Acute Liver Failure. Digest. Dis. Sci. 2010, 55, 285-291. (30) Yu, Q.; Liu, L.; Lin, J.; Wang, Y.; Xuan, X.; Guo, Y.; Hu, S. SDF-1α/CXCR4 Axis Mediates The Migration of Mesenchymal Stem Cells to The Hypoxic-Ischemic Brain Lesion in A Rat Model. Cell Journal (Yakhteh) 2015, 16, 440-447. 23



(31) Tuljapurkar, S. R.; McGuire, T. R.; Brusnahan, S. K.; Jackson, J. D.; Garvin, K. L.; Kessinger, M. A.; Lane, J. T.; BJ, O. K.; Sharp, J. G. Changes in Human Bone Marrow Fat Content Associated with Changes in Hematopoietic Stem Cell Numbers and Cytokine Levels with Aging. J. Anat. 2011, 219, 574-581. (32) Lien, C. Y.; Ho, K. C. Y.; Lee, O. K.; Blunn, G. W.; Su, Y. Restoration of Bone Mass and Strength in Glucocorticoid-Treated Mice by Systemic Transplantation of CXCR4 and Cbfa-1 Co-Expressing Mesenchymal Stem Cells. J. Bone Miner. Res. 2009, 24, 837-848. (33) Ryu, T. K.; Kang, R. H.; Jeong, K. Y.; Jun, D. R.; Koh, J. M.; Kim, D.; Bae, S. K.; Choi, S. W. Bone-Targeted Delivery of Nanodiamond-Based Drug Carriers Conjugated with Alendronate for Potential Osteoporosis Treatment. J. Controlled Release 2016, 232, 152-160. (34) Chen, H.; Li, G.; Chi, H.; Wang, D.; Tu, C.; Pan, L.; Zhu, L.; Qiu, F.; Guo, F.; Zhu, X. Alendronate-Conjugated Amphiphilic Hyperbranched Polymer Based on Boltorn H40 and Poly(ethylene glycol) for Bone-targeted Drug Delivery. Bioconjug. Chem. 2012, 23, 1915-1924. (35) Clementi, C.; Miller, K.; Mero, A.; Satchi-Fainaro, R.; Pasut, G. Dendritic Poly(ethylene glycol) Bearing Paclitaxel and Alendronate for Targeting Bone Neoplasms. Mol. Pharm. 2011, 8, 1063-1072. (36) Thamake, S. I.; Raut, S. L.; Gryczynski, Z.; Ranjan, A. P.; Vishwanatha, J. K. Alendronate Coated Poly-Lactic-co-Glycolic Acid (PLGA) Nanoparticles for Active Targeting of Metastatic Breast Cancer. Biomaterials 2012, 33, 7164-7173. (37) 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. (38) Balazs, D. A.; Godbey, W. Liposomes for Use in Gene Delivery. J. Drug Deliv. 2011, 2011,326497. (39) Noble, G. T.; Stefanick, J. F.; Ashley, J. D.; Kiziltepe, T.; Bilgicer, B. Ligand-Targeted Liposome Design: Challenges and Fundamental Considerations. Trends Biotechnol. 2014, 32, 32-45. (40) Nguyen, T. D.; Pitchaimani, A.; Aryal, S. Engineered Nanomedicine with Alendronic Acid Corona Improves Targeting to Osteosarcoma. Sci. Rep. 2016, 6, 36707. (41) Zhang, G.; Guo, B.; Wu, H.; Tang, T.; Zhang, B. T.; Zheng, L.; He, Y.; Yang, Z.; Pan, X.; Chow, H.; To, K.; Li, Y.; Li, D.; Wang, X.; Wang, Y.; Lee, K.; Hou, Z.; Dong, N.; Li, G.; Leung, K.; Hung, L.; He, F.; Zhang, L.; Qin, L. A Delivery System Targeting Bone Formation Surfaces to Facilitate RNAi-Based Anabolic Therapy. Nat. Med. 2012, 18, 307-314. 24


Page 24 of 39

Page 25 of 39 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


(42) Morelli, S.; Bilbao, P. S.; Katz, S.; Lezcano, V.; Roldan, E.; Boland, R.; Santillan, G. Protein Phosphatases: Possible Bisphosphonate Binding Sites Mediating Stimulation of Osteoblast Proliferation. Arch. Biochem. Biophys. 2011, 507, 248-253. (43) Srinivas, R.; Samanta, S.; Chaudhuri, A. Cationic Amphiphiles: Promising Carriers of Genetic Materials in Gene Therapy. Chem. Soc. Rev. 2009, 38, 3326-3338. (44) Lee, Y.; Koo, H.; Lim, Y.-b.; Lee, Y.; Mo, H.; Sang Park, J. New Cationic Lipids for Gene Transfer with High Efficiency and Low Toxicity: T-Shape Cholesterol Ester Derivatives. Bioorg. Med. Chem. Lett. 2004, 14, 2637-2641. (45) Lapidot, T.; Dar, A.; Kollet, O. How Do Stem Cells Find Their Way Home? Blood 2005, 106, 1901-1910. (46) Lapidot, T.; Petit, I. Current Understanding of Stem Cell Mobilization: the Roles of Chemokines, Proteolytic Enzymes, Adhesion Molecules, Cytokines, and Stromal Cells. Exp. Hematol. 2002, 30, 973-981. (47) Frangogiannis, N. G.; Entman, M. L. Chemokines in Myocardial Ischemia. Trends Cardiovasc. Med. 2005, 15, 163-169. (48) Kitaori, T.; Ito, H.; Schwarz, E. A.; Tsutsumi, R.; Yoshitomi, H.; Oishi, S.; Nakano, M.; Fujii, N.; Nagasawa, T.; Nakamura, T. Stromal Cell-Derived Factor 1/CXCR4 Signaling Is Critical for the Recruitment of Mesenchymal Stem Cells to the Fracture Site During Skeletal Repair in A Mouse Model. Arthritis Rheum-Us 2009, 60, 813-823. (49) Wynn, R. F.; Hart, C. A.; Corradi-Perini, C.; O'Neill, L.; Evans, C. A.; Wraith, J. E.; Fairbairn, L. J.; Bellantuono, I. A Small Proportion of Mesenchymal Stem Cells Strongly Expresses Functionally Active CXCR4 Receptor Capable of Promoting Migration to Bone Marrow. Blood 2004, 104, 2643-2645. (50) Patrulea, V.; Ostafe, V.; Borchard, G.; Jordan, O. Chitosan as A Starting Material for Wound Healing Applications. Eur. J. Pharm. Biopharm. 2015, 97, 417-426. (51) Kanasty, R.; Dorkin, J. R.; Vegas, A.; Anderson, D. Delivery Materials for SiRNA Therapeutics. Nat. Mater. 2013, 12, 967-977. (52) Blanco, E.; Shen, H.; Ferrari, M. Principles of Nanoparticle Design for Overcoming Biological Barriers to Drug Delivery. Nat. Biotechnol. 2015, 33, 941-951. (53) Sun, Y.; Ye, X. Z.; Cai, M. X.; Liu, X. N.; Xiao, J.; Zhang, C. Y.; Wang, Y. Y.; Yang, L.; Liu, J. F.; 25



Page 26 of 39

Li, S. N.; Kang, C.; Zhang, B.; Zhang, Q.; Wang, Z. L.; Hong, A.; Wang, X. G. Osteoblast-Targeting-Peptide Modified Nanoparticle for siRNA/microRNA Delivery. Acs Nano 2016, 10, 5759-5768. (54) Liang, C.; Guo, B.; Wu, H.; Shao, N.; Li, D.; Liu, J.; Dang, L.; Wang, C.; Li, H.; Li, S.; Lau, W. K.; Cao, Y.; Yang, Z.; Lu, C.; He, X.; Au, D. W.; Pan, X.; Zhang, B. T.; Lu, C.; Zhang, H.; Yue, K.; Qian, A.; Shang, P.; Xu, J.; Xiao, L.; Bian, Z.; Tan, W.; Liang, Z.; He, F.; Zhang, L.; Lu, A.; Zhang, G. Aptamer-Functionalized

Lipid

Nanoparticles

Targeting

Osteoblasts

as

A

Novel

RNA

Interference-Based Bone Anabolic strategy. Nat. Med. 2015, 21, 288-294. (55) Wang, Y. C.; Newman, M. R.; Ackun-Farmmer, M.; Baranello, M. P.; Sheu, T. J.; Puzas, J. E.; Benoit, D. S. W. Fracture-Targeted Delivery of beta-Catenin Agonists via Peptide-Functionalized Nanoparticles Augments Fracture Healing. Acs Nano 2017, 11, 9445-9458. (56) Low, S. A.; Galliford, C. V.; Yang, J.; Low, P. S.; Kopecek, J. Biodistribution of Fracture-Targeted

GSK3beta

Inhibitor-Loaded

Micelles

for

Improved

Fracture

Healing.

Biomacromolecules 2015, 16, 3145-3153. (57) Liu, J.; Dang, L.; Li, D.; Liang, C.; He, X.; Wu, H.; Qian, A.; Yang, Z.; Au, D. W.; Chiang, M. W.; Zhang, B. T.; Han, Q.; Yue, K. K.; Zhang, H.; Lv, C.; Pan, X.; Xu, J.; Bian, Z.; Shang, P.; Tan, W.; Liang, Z.; Guo, B.; Lu, A.; Zhang, G. A Delivery System Specifically Approaching Bone Resorption Surfaces to Facilitate Therapeutic Modulation of MicroRNAs in Osteoclasts. Biomaterials 2015, 52, 148-160. (58) 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. Osteoporos. Int. 2008, 19, 733-759. (59) Ebetino, F. H.; Hogan, A. M. L.; Sun, S. T.; Tsoumpra, M. K.; Duan, X. C.; Triffitt, J. T.; Kwaasi, A. A.; Dunford, J. E.; Barnett, B. L.; Oppermann, U.; Lundy, M. W.; Boyde, A.; Kashemirov, B. A.; McKenna, C. E.; Russell, R. G. G. The Relationship Between the Chemistry and Biological Activity of the Bisphosphonates. Bone 2011, 49, 20-33. (60) Butcher, E. C. Leukocyte-Endothelial Cell Recognition: Three (or more) Steps to Specificity and Diversity. Cell 1991, 67, 1033-1036. (61) Laird, D. J.; von Andrian, U. H.; Wagers, A. J. Stem Cell Trafficking in Tissue Development, Growth, and Disease. Cell 2008, 132, 612-630. 26


Page 27 of 39 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


(62) Ruster, B.; Gottig, S.; Ludwig, R. J.; Bistrian, R.; Muller, S.; Seifried, E.; Gille, J.; Henschler, R. Mesenchymal Stem Cells Display Coordinated Rolling and Adhesion Behavior on Endothelial Cells. Blood 2006, 108, 3938-3944. (63) Schmidt, A.; Ladage, D.; Steingen, C.; Brixius, K.; Schinkothe, T.; Klinz, F. J.; Schwinger, R. H.; Mehlhorn, U.; Bloch, W. Mesenchymal Stem Cells Transmigrate over the Endothelial Barrier. Eur. J. Cell Biol. 2006, 85, 1179-1188. (64) Swami, A.; Reagan, M. R.; Basto, P.; Mishima, Y.; Kamaly, N.; Glavey, S.; Zhang, S.; Moschetta, M.; Seevaratnam, D.; Zhang, Y.; Liu, J.; Memarzadeh, M.; Wu, J.; Manier, S.; Shi, J.; Bertrand, N.; Lu, Z. N.; Nagano, K.; Baron, R.; Sacco, A.; Roccaro, A. M.; Farokhzad, O. C.; Ghobrial, I. M. Engineered Nanomedicine for Myeloma and Bone Microenvironment Targeting. Proc. Natl. Acad. Sci .U .S .A. 2014, 111, 10287-10292. (65) Jones, C. H.; Chen, C.-K.; Ravikrishnan, A.; Rane, S.; Pfeifer, B. A. Overcoming Nonviral Gene Delivery Barriers: Perspective and Future. Mol. Pharm. 2013, 10, 4082-4098. (66) Pignatello, R.; Sarpietro, M. G.; Castelli, F. Synthesis and Biological Evaluation of A New Polymeric Conjugate and Nanocarrier with Osteotropic Properties. J. Funct. Biomater. 2012, 3, 79-99. (67) Sun, X.; Pang, Z.; Ye, H.; Qiu, B.; Guo, L.; Li, J.; Ren, J.; Qian, Y.; Zhang, Q.; Chen, J.; Jiang, X. Co-Delivery of pEGFP-hTRAIL and Paclitaxel to Brain Glioma Mediated by An Angiopep-Conjugated Liposome. Biomaterials 2012, 33, 916-924. (68) Inoh, Y.; Nagai, M.; Matsushita, K.; Nakanishi, M.; Furuno, T. Gene Transfection Efficiency into Dendritic Cells is Influenced by the Size of Cationic Liposomes/DNA Complexes. Eur. J. Pharm. Sci. 2017, 102, 230-236. (69) Tang, Y.; Wu, X.; Lei, W.; Pang, L.; Wan, C.; Shi, Z.; Zhao, L.; Nagy, T. R.; Peng, X.; Hu, J.; Feng, X.; Van Hul, W.; Wan, M.; Cao, X. TGF-Beta1-Induced Migration of Bone Mesenchymal Stem Cells Couples Bone Resorption with Formation. Nat. Med. 2009, 15, 757-765.

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Figure captions: Figure 1. Preparation of alendronate functionalized liposomes (Aln-Lipo). (A) Synthesis of DSPE-PEG-Aln via carbodiimide reaction. (B) Schematic depiction of Aln modified liposomes prepared by thin-film hydration. (C) Chemical structure characterization by 1H NMR with Aln in D2O and DSPE-PEG-Aln in CDCl3 confirmed the formation of DSPE-PEG-Aln conjugate.

Figure 2. Characterization of alendronate functionalized liposomes (Aln-Lipo). (A) Hydrodynamic diameter and zeta-potential of liposomes with and without Aln modification dispersed in PBS, n=3. (B) Representative TEM images of Aln-Lipo-DNA stained with phosphotungstic acid. (C) Gel electrograms (agarose, 0.8%) of Aln-Lipo-DNA at various N/P ratios (Aln-Lipo: DNA, from 0:1 to 4:1) to study the DNA condensation ability of liposome. (D) The DNA encapsulation efficiency of Aln-Lipo at various N/P ratios (Aln-Lipo: DNA, from 1:1 to 8:1) by ethidium bromide assay. (E) The binding affinity of NBD-labeled Lipo and Aln-Lipo to hydroxyapatite (HAp) by fluorescence detection, n=3. ***p < 0.001 indicates significance evaluated using Student’s t-test.

Figure 3. Transfection efficiency and cytotoxicity characterization in vitro. (A) Transfection efficiency was formulated on COS-1 cells by luciferase expression, n=3. (B) Comparison of GFP expression between the liposomes with and without alendronate modification on COS-1 and MC3T3-E1 cells, and the results were quantified by flow cytometry, n=3. Scale bar: 100 μm. (C) The cell viability of liposomes was investigated on MC3T3-E1 osteoblast cells by CCK-8 assay, n = 6. ***p < 0.001 indicates significance, n.s. indicates not significant. One-way ANOVA 28


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with a Tukey’s post-hoc test was performed.

Figure 4. The attracting effect of expressed SDF-1 on MSCs migration investigated in vitro. (A) The SDF-1 mRNA expression in transfected MC3T3-E1 osteoblastic cells was studied by qRT-PCR 24 h post-transfection, n=3. (B) Representative Western blot analysis of SDF-1 protein expression from MC3T3-E1 cells 48 h post-transfection. (C) Schematic depiction of transwell assay in studying MSCs migration, Ob:MC3T3-E1 osteoblastic cells. (D) The migrated C3H10T1/2 stem cells in the lower surface of transwell membrane attracted by the expressed SDF-1 from transfected MC3T3-E1 osteoblastic cells in the lower chamber was stained by crystal violet. (E) Quantification of the migrated C3H10T1/2 by dissolving crystal violet and spectrophotometrically measured at 573 nm, n=3, the resulted optical density (OD) was normalized by control. Plasmid pCMV-SDF-1 alone was used as control, *p < 0.05 and ***p < 0.001 indicate significance evaluated using One-way ANOVA with a Tukey’s post-hoc test.

Figure 5. Bone targeted SDF-1 gene delivery and inducing MSCs migration. (A) Schematic diagram illustrating the experimental design. Liposomes with SDF-1 gene and GFP+ MSCs were intravenously administrated, and the biodistribution of delivered gene and cells as well as the bone regeneration were investigated. (B) Biodistribution of EMA-labeled SDF-1 gene in major organs (heart, liver, spleen, lung, kidney and femur) was visualized by biophotonic imaging at 6 h after treatment on Xenogen IVIS Imaging System, n=3. (C) GFP+ MSCs in bone marrow of femurs by immunofluorescent staining (scale bar 20 μm, n=3) and (D) quantified by flow 29



cytometry for the normalized percentage of GFP+ MSCs in bone marrow cell (n = 5). (E) Immunofluorescent staining showed the accumulation of GFP+ MSCs around vessel. (F) Representative images showing three-dimensional trabecular architecture by micro-CT reconstruction in the distal femurs and lumbar vertebra l(scale bar 1 mm). (G) The structure parameters of trabecular bone calculated after three-dimension reconstruction: bone mineral density (BMD), bone volume/tissue volume (BV/TV), Tb.Th (trabecular thickness) and Tb.N (trabecular numbers), n = 5. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate significance evaluated using One-way ANOVA with a Tukey’s post-hoc test.

Figure 6. Schematic representation of alendronate functionalized nanoparticles for bone targeted gene delivery of SDF-1 in attraction of stem cell homing and participating in bone regeneration. Alendronate facilitated the targeting of Aln-Lipo-SDF-1 nanoparticles to bone by the chelating interaction between bisphosphonates of alendronate and calcium ion in bone mineral. The transfected osteoblast on bone surface could secrete SDF-1 protein into bone marrow and form the concentration gradient. The stem cells could response to SDF-1 gradient and home to the bone surface. Influenced by the microenvironment of bone surface, stem cells different into osteoblast and contribute to the bone regeneration.

Table 1. The effect of Aln-Lipo-SDF-1 treatment on the microarchitecture of trabecular bone in femur and vertebrae analyzed by Micro-CT.

30


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Figure 1

Figure 1. Preparation of alendronate functionalized liposomes (Aln-Lipo). (A) Synthesis of DSPE-PEG-Aln via carbodiimide reaction. (B) Schematic depiction of Aln modified liposomes prepared by thin-film hydration. (C) Chemical structure characterization by 1H NMR with Aln in D2O and DSPE-PEG-Aln in CDCl3 confirmed the formation of DSPE-PEG-Aln conjugate.

31



Figure 2

Figure 2. Characterization of alendronate functionalized liposomes (Aln-Lipo). (A) Hydrodynamic diameter and zeta-potential of liposomes with and without Aln modification dispersed in PBS, n=3. (B) Representative TEM images of Aln-Lipo-DNA stained with phosphotungstic acid. (C) Gel electrograms (agarose, 0.8%) of Aln-Lipo-DNA at various N/P ratios (Aln-Lipo: DNA, from 0:1 to 4:1) to study the DNA condensation ability of liposome. (D) The DNA encapsulation efficiency of Aln-Lipo at various N/P ratios (Aln-Lipo: DNA, from 1:1 to 8:1) by ethidium bromide assay. (E) The binding affinity of NBD-labeled Lipo and Aln-Lipo to hydroxyapatite (HAp) by fluorescence detection, n=3. ***p < 0.001 indicates significance evaluated using Student’s t-test. 32


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Figure 3

Figure 3. Transfection efficiency and cytotoxicity characterization in vitro. (A) Transfection efficiency was formulated on COS-1 cells by luciferase expression, n=3. (B) Comparison of GFP expression between the liposomes with and without alendronate modification on COS-1 and MC3T3-E1 cells, and the results were quantified by flow cytometry, n=3. Scale bar: 100 μm. (C) The cell viability of liposomes was investigated on MC3T3-E1 osteoblast cells by CCK-8 assay, n = 6. ***p < 0.001 indicates significance, n.s. indicates not significant. One-way ANOVA with a Tukey’s post-hoc test was performed.

33



Figure 4

Figure 4. The attracting effect of expressed SDF-1 on MSCs migration investigated in vitro. (A) The SDF-1 mRNA expression in transfected MC3T3-E1 osteoblastic cells was studied by qRT-PCR 24 h post-transfection, n=3. (B) Representative Western blot analysis of SDF-1 protein expression from MC3T3-E1 cells 48 h post-transfection. (C) Schematic depiction of transwell assay in studying MSCs migration, Ob:MC3T3-E1 osteoblastic cells. (D) The migrated C3H10T1/2 stem cells in the lower surface of transwell membrane attracted by the expressed SDF-1 from transfected MC3T3-E1 osteoblastic cells in the lower chamber was stained by crystal violet. (E) Quantification of the migrated C3H10T1/2 by dissolving crystal violet and spectrophotometrically measured at 573 nm, n=3, the resulted optical density (OD) was normalized by control. Plasmid pCMV-SDF-1 alone was used as control, *p < 34


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0.05 and ***p < 0.001 indicate significance evaluated using One-way ANOVA with a Tukey’s post-hoc test.

Figure 5

Figure 5. Bone targeted SDF-1 gene delivery and inducing MSCs migration. (A) Schematic diagram illustrating the experimental design. Liposomes with SDF-1 gene 35



and GFP+ MSCs were intravenously administrated, and the biodistribution of delivered gene and cells as well as the bone regeneration were investigated. (B) Biodistribution of EMA-labeled SDF-1 gene in major organs (heart, liver, spleen, lung, kidney and femur) was visualized by biophotonic imaging at 6 h after treatment on Xenogen IVIS Imaging System, n=3. (C) GFP+ MSCs in bone marrow of femurs by immunofluorescent staining (scale bar 20 μm, n=3) and (D) quantified by flow cytometry for the normalized percentage of GFP+ MSCs in bone marrow cell (n = 5). (E) Immunofluorescent staining showed the accumulation of GFP+ MSCs around vessel. (F) Representative images showing three-dimensional trabecular architecture by micro-CT reconstruction in the distal femurs and lumbar vertebral (scale bar 1 mm). (G) The structure parameters of trabecular bone calculated after three-dimension reconstruction: bone mineral density (BMD), bone volume/tissue volume (BV/TV), Tb.Th (trabecular thickness) and Tb.N (trabecular numbers), n = 5. *p < 0.05, **p < 0.01 and ***p < 0.001 indicate significance evaluated using One-way ANOVA with a Tukey’s post-hoc test.

36


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Figure 6

Figure 6. Schematic representation of alendronate functionalized nanoparticles for bone targeted gene delivery of SDF-1 in attraction of stem cell homing and participating in bone regeneration. Alendronate facilitated the targeting of Aln-Lipo-SDF-1 nanoparticles to bone by the chelating interaction between bisphosphonates of alendronate and calcium ion in bone mineral. The transfected osteoblast on bone surface could secrete SDF-1 protein into bone marrow and form the concentration gradient. The stem cells could response to SDF-1 gradient and home to the bone surface. Influenced by the microenvironment of bone surface, stem cells different into osteoblast and contribute to the bone regeneration.

37



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Table 1. The effect of Aln-Lipo-SDF-1 treatment on the microarchitecture of trabecular bone in femur and vertebrae analyzed by Micro-CT

Group

BMD

BV/TV

Tb.Th

Tb.N

(g/cm3)

(%)

(mm)

(/mm)

Femur-C

0.528±0.089 12.6±1.8

0.018±0.001 7.19±1.24

Femur-T

0.701±0.049 17.8±1.6

0.023±0.003 7.98±1.61

Vertebrae-C 0.459±0.065 12.0±1.3

0.017±0.003 7.13±0.73

Vertebrae-T

0.021±0.001 9.48±1.43

0.709±0.101 19.3±1.6

Contro vs. AL-SDF-1, One-way ANOVA, p Femur

0.015

0.001

0.016

n.s.

Vertebrae

0.001

＜0.001

n.s.

0.049

BMD = bone mineral density, BV/TV = bone volume/tissue volume, Tb.Th = trabecular thickness, Tb.N = trabecular numbers, C = control, T= treatment with AS-SDF-1, n.s.= not significant.

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Table of Contents

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Bone Targeted Delivery of SDF-1 via Alendronate Functionalized

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