Research Article Cite This: ACS Appl. Mater. Interfaces 2018, 10, 23700−23710
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Bone Targeted Delivery of SDF‑1 via Alendronate Functionalized Nanoparticles in Guiding Stem Cell Migration Qingchang Chen,†,# Chuping Zheng,†,§,# Yanqun Li,†,# Shaoquan Bian,† Haobo Pan,† Xiaoli Zhao,*,† and William W. Lu‡
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†
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 ‡ Department of Orthopaedic and Traumatology, The University of Hong Kong, 21 Sassoon Rd., Pokfulam, 999077, Hong Kong, PR China § School of Pharmaceutical Science, Guangzhou Medical University, Guangzhou, Guangdong, 511436, PR China S Supporting Information *
ABSTRACT: Stem cells are well-known for their great capacity for 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 cell 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. This study may provide a useful technique in regulating stem cell migration. KEYWORDS: bone targeting, gene delivery, stem cell homing, SDF-1, osteoporosis therapy
1. INTRODUCTION Osteoporosis is a worldwide health problem related to the aging population.1 Unbalanced 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 their great capacity for tissue regeneration.7,8 Because of the osteogenic potential as well as ease of isolation and expansion, they have become a promising source for cell-based therapy in treating various bone degenerative disorders.9,10 Systemic MSC 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 have not been observed thus far.11,12 Poor bone marrow homing and low engraftment efficiencies are the major hurdles for systemic infusion of MSCs. © 2018 American Chemical Society
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 MSCs. Increasing evidence suggest that MSCs could home to damaged tissues in response to injury.18−20 It is 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 cell homing.22 It has been found up-regulated at injury sites and served as a potent Received: May 24, 2018 Accepted: June 25, 2018 Published: June 25, 2018 23700
DOI: 10.1021/acsami.8b08606 ACS Appl. Mater. Interfaces 2018, 10, 23700−23710
Research Article
ACS Applied Materials & Interfaces
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.
2. RESULTS AND DISCUSSION 2.1. Preparation of Alendronate Functionalized Liposomes. Nonviral vectors mediated systemic gene delivery has many advantages, particularly with respect to safety.37 Lipid-based gene carriers are among the most widely used nonviral 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 the 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
chemoattractant to recruit MSCs. The transiently expressed SDF-1 in ischemic cardiomyopathy model showed the attraction of stem cells to injured myocardium.23 In ischemic heart failure model, overexpression of SDF-1 in myocardium could recruit endogenous cardiac stem cells and promote 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 to be 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 cell homing, bone targeted nanoparticles carrying the SDF-1 gene was developed in this study and expected to enhance stem cell 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. 23701
DOI: 10.1021/acsami.8b08606 ACS Appl. Mater. Interfaces 2018, 10, 23700−23710
Research Article
ACS Applied Materials & Interfaces
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) 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) 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 MC3T3E1 cells, and the results were quantified by flow cytometry, n = 3. Scale bar: 100 μm. (C) 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.
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DOI: 10.1021/acsami.8b08606 ACS Appl. Mater. Interfaces 2018, 10, 23700−23710
Research Article
ACS Applied Materials & Interfaces
Figure 4. Attracting effect of expressed SDF-1 on MSCs migration investigated in vitro. (A) 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) Migrated C3H10T1/2 stem cells in the lower surface of transwell membrane attracted by the expressed SDF-1 from transfected MC3T3E1 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.
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 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 Lipofectamine 2000 (Lipo2000). Aln modification did not have influence on efficiency in these cells. This was further studied by GFP gene transfection and 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 MC3T3E1 cells from 15.1% to 24.4%, whereas the increment in COS-1 cells was not as 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 receptors.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. 23703
DOI: 10.1021/acsami.8b08606 ACS Appl. Mater. Interfaces 2018, 10, 23700−23710
Research Article
ACS Applied Materials & Interfaces
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 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) 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.
2.4. In Vitro MSCs Migration. Stem cells provide a promising approach 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 (SDF1) has been demonstrated with important role in stem cell homing. It was observed to be 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
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. 23704
DOI: 10.1021/acsami.8b08606 ACS Appl. Mater. Interfaces 2018, 10, 23700−23710
Research Article
ACS Applied Materials & Interfaces
Table 1. Effect of Aln-Lipo-SDF-1 Treatment on the Microarchitecture of Trabecular Bone in Femur and Vertebrae Analyzed by Micro-CTa Group
BMD (g/cm3)
Femur-C 0.528 ± 0.089 Femur-T 0.701 ± 0.049 Vertebrae-C 0.459 ± 0.065 Vertebrae-T 0.709 ± 0.101 Contro vs AL-SDF-1, One-Way ANOVA, p Femur 0.015 Vertebrae 0.001
BV/TV (%) 12.6 17.8 12.0 19.3
± ± ± ±
1.8 1.6 1.3 1.6
0.001
